Patent Application
20240016391
The present application relates generally to the fields of detection and elimination of malaria parasites in a patient's body, in particular, with the use of laser-induced transient vapor nanobubbles.
Malaria is a widespread and infectious disease that can cause serious illness and death in humans. A patient can be infected when a malaria parasite infects cells of the patient, also known as a host. The parasite can produce hemozoin (HZ), which are nanocrystals formed when the parasite digests the hemoglobin in the host's red blood cells. Malaria-infected red blood cells or other body tissue infected by malaria parasites contain HZ (hemozoin) nanocrystals.
Current malaria diagnosis techniques include, for example, rapid diagnostic tests (RDTs), microscopy, and polymerase chain reaction (PCR). These diagnosis techniques analyze a patient's blood samples. RDT analyzes the proteins in the blood to look for presence of malaria parasites and is approved by the World Health Organization (WHO). Microscopy uses stain of a thick blood slide, such as with a 200 to 500 white blood cell count, to determine malaria parasite density and gametocyte counts. Microscopy is also WHO-approved for malaria diagnosis. PCR analyzes DNAs in the blood to determine presence of malaria parasites.
Malaria can be treated and/or prevented by administration of antimalarial drugs, such as quinine, chloroquine, atovaquone/proguanil, and others.
Current malaria diagnosis generally employ invasive techniques which are costly, time-consuming and have low accuracy. The diagnosis and treatment of malaria can require separate procedures.
Antimalarial drugs have several disadvantages. Malaria parasites can develop drug resistance to the antimalarial drugs. The drugs can also be ineffective against malaria parasites that escape from the blood vessels into the tissue and/or skin of the patient through micro-capillaries in the tissue, also known as tissue-sequestered parasites. Tissue-sequestered parasites can cause lethal complications in the patient after treatments with antimalarial drugs, such as when the patient has low blood levels of parasites, and/or can cause relapses in patients treated with antimalarial drugs. Current malaria diagnosis techniques, such as RDTs, microscopy, and PCR, are not able to detect tissue-sequestered malaria parasites as these techniques rely on analyzing the patient's peripheral blood samples.
Current malaria diagnosis techniques also may not detect the HZ (hemozoin) nanocrystals without an active and/or live malaria parasite. However, detecting the HZ (hemozoin) nanocrystals without an active or live malaria parasite can provide valuable information for determining recent or past presence of malaria infection, the data important in screening and understanding the malaria transmission.
Laser-induced transient vapor nanobubbles can be used to diagnose and/or treat malaria in a noninvasive, efficient, and reproducible manner. The transient vapor nanobubbles can be generated around one or more malaria-specific nanoparticles (such as one or more HZ (hemozoin) nanocrystals (with or without an active malaria parasite) or malaria-specific nanoparticles introduced into the host red blood cells) when laser pulses are applied to the nanoparticles. The laser pulses can cause rapid heating of the malaria-specific nanoparticles, but not of uninfected red blood cells or other host tissues. Liquid (such as water) around the malaria-specific nanoparticles can rapidly evaporate, leading to the generation of a transient vapor nanobubble. The generation of transient vapor nanobubbles can be detected by acoustic detectors. In some embodiments, the transient nanobubble-based malaria detection mechanism can detect a single hemozoin nanoparticle. The transient nanobubble-based malaria detection mechanism disclosed herein can be advantageous over the bulk photoacoustic mechanism, which requires a large number of hemozoin nanoparticles to produce a detectable malaria-positive signal.
As the transient vapor nanobubble size increases with increasing energy level of the laser pulses, in some instances, the energy level of the laser pulses can be high enough to generate transient vapor nanobubbles that can cause mechanical damage to the HZ (hemozoin) nanocrystal host, the malaria parasite, the malaria-infected red blood cell, or a combination thereof. Additional details of employing transient vapor nanobubbles to detect and/or treat malaria-infected red blood cells are described in International Application No. WO2013/109722, filed Jan. 17, 2013 and titled “Theranostic methods and systems for diagnosis and treatment of malaria,” attached as Appendix A, the entirety of which is incorporated herein by reference and should be considered a part of the specification.
The vapor nanobubbles, also referred to as HZ (hemozoin)-generated vapor nanobubbles (HVNB) or nanobubbles, can be generated on a liquid sample test (such as blood, in particular peripheral blood, or urine), or on a patient's skin using a sensor that optically excites the malaria-specific nanoparticle in the skin to generate hemozoin-generated vapor nanobubbles.
In order to use transient vapor nanobubbles for detecting and/or treating malaria noninvasively, the laser pulses must penetrate a patient's skin and reach the malaria-specific nanoparticles despite attenuation of the laser pulses by the patient's body tissue as the laser pulses travel deeper under the skin. The optical delivery part of the malaria sensor, such as an optical fiber, needs to be brought as close as possible to the malaria-specific nanoparticles or malaria parasite. The nanobubble-generated pressure pulses reaching the surface of the acoustic detector also need to be strong enough for an acoustic (ultrasound) signal of that pressure pulse to be detected. Challenges in improving the sensitivity and/or specificity of the acoustic detector can include reducing a distance between the malaria-specific nanoparticles and the acoustic detector, and/or reducing a distance between an optical source and the acoustic detector.
In some instances, a malaria diagnosis and/or treatment apparatus can combine an optical source and an acoustic detector in a single probe (also referred to as a malaria sensor). The single probe can bring the acoustic detector closer to the source of the acoustic pulse, which is the transient vapor nanobubble, than having separate optical source and acoustic detector probes. However, the distance between the acoustic detector surface and the malaria-specific nanoparticle in the known single probes can still be too large. This can be due to the use of a spherical acoustic detector for detecting the transient vapor nanobubble, as the transient vapor nanobubble is considered a point source. The spherical acoustic detector can also have a large surface area, which weakens the signal output from the spherical detector. Other concerns with using a spherical acoustic detector can include high cost and structural complexity, which can make it infeasible to mass-produce the single probe.
The malaria probe according to the present disclosure can include the optical source and the acoustic detector in a single probe, and can diagnose and/or treat malaria. The malaria probe according to the present disclosure can increase the sensitivity and specificity of the malaria detection by having one or more small and substantially flat acoustic detectors placed in close proximity to the optical source, which can include one or more optical fibers, and to a probe tip surface. Sensitivity can be indicative of the probe's ability to correctly detect malaria-positive cases. Specificity can be indicative of the probe's ability to avoid false positive and false negative detections. In some embodiments, the malaria probe is able to detect tissue-sequestered malaria parasites. Embodiments of the malaria probe can also be immune to resistance from the malaria parasite, efficient, and/or safe for the patient. In some embodiments, the malaria probe can be cheap to build and/or economically feasible for mass production.
An apparatus configured for diagnosing malaria noninvasively can comprise: a sensor probe having a probe body terminating at a probe tip surface, the probe tip surface configured to be placed into contact with a predetermined detection location, the predetermined detection location being in vivo on a patient's skin or ex vivo in a patient's body fluid sample; an optical source configured to generate a plurality of laser pulses of at least one predetermined energy level or at least one predetermined wavelength, the optical source terminating at or near the probe tip surface, the laser pulses configured to cause generation of one or more transient vapor nanobubbles around malaria-specific nanoparticles at the predetermined detection location; and one or more acoustic detectors configured to detect acoustic pulses generated by the one or more transient vapor nanobubbles and output one or more signals indicative of the detected acoustic pulses to at least one processor, the one or more acoustic detectors comprising a piezo element and being flat, wherein a distance between an outer wall of the optical source and a radially inner edge of the one or more acoustic detectors, R1, can be 0.01 mm to 0.03 mm so as to improve a signal strength of the acoustic pulses striking a flat surface of the one or more acoustic detectors, wherein the R1 can be 0.01 mm to 0.03 mm such that the acoustic pulses strike the flat surface of the one or more acoustic detectors at an angle of incidence, a, of less than 45°, wherein the optical source and the one or more acoustic detectors can be enclosed within the probe body.
In a configuration, the optical source can comprise one or more optical fibers.
In a configuration, the one or more optical fibers can each have a core diameter of about 100 μm.
In a configuration, the piezo element can comprise a navy type II or type VI material or a composite material.
In a configuration, a tissue-facing surface of the one or more acoustic detectors can be 0.1 mm to 0.3 mm recessed from the probe tip surface.
In a configuration, the apparatus can further comprise a front layer between the probe tip surface and a tissue-facing surface of the one or more acoustic detectors.
In a configuration, an outer surface of the one or more optical fibers can be separated from a radially outer edge of the one or more acoustic detectors by 0.3 mm to 1.5 mm.
In a configuration, the malaria-specific nanoparticles can be located within an optical penetration depth beneath the predetermination location.
In a configuration, an outer diameter of the one or more acoustic detectors can be 0.2 mm to 3 mm such that the acoustic pulses strike the flat surface of the one or more acoustic detectors at a same or similar angle of incidence to reduce an effect of de-phasing.
An apparatus configured for diagnosing malaria noninvasively can comprise: a sensor portion, the sensor portion including an optical source configured to generate laser pulses of at least one predetermined energy level, the optical source comprising an optical fiber terminating at or near a distal end of the sensor portion, the laser pulses configured to cause generation of one or more nanobubbles around malaria-specific nanoparticles at a predetermined location, and one or more acoustic detectors configured to detect acoustic pulses generated by the one or more nanobubbles and output one or more signals indicative of the detected acoustic pulses to at least one processor, the one or more acoustic detectors comprising a piezo element and being flat, wherein, at the distal end of the sensor portion, a distance between an outer wall of the optical fiber and a radially inner edge of the one or more acoustic detectors, R1, can be 0.01 mm to 0.03 mm so as to improve a signal strength of the acoustic pulses striking a flat surface of the one or more acoustic detectors, wherein the R1 can be 0.01 mm to 0.03 mm such that the acoustic pulses strike the flat surface of the one or more acoustic detectors at an angle of incidence, a, of less than 45°; a housing, wherein the sensor portion can be at least partially disposed within the housing; and a spring disposed between a proximal end of the housing and the proximal end of the sensor portion, the spring biasing the sensor portion toward a distal end of the housing.
In a configuration, the spring can be configured to be compressed when the apparatus is applied to a measurement site, the compressed spring forcing the distal end of the sensor portion into contact with the measurement site.
In a configuration, the housing can comprise a patient interface at the distal end, the apparatus further comprising a liner covering the patient interface when the apparatus is not in use.
In a configuration, the patient interface can comprise an adhesive layer and/or a gel layer.
In a configuration, an outer diameter of the one or more acoustic detectors can be 0.2 mm to 3 mm such that the acoustic pulses strike the flat surface of the one or more acoustic detectors at a same or similar angle of incidence to reduce an effect of de-phasing.
An apparatus for diagnosing and/or treating malaria in a patient noninvasively can comprise a sensor probe having a probe body terminating at a probe tip surface, the probe tip surface configured to be placed into contact with a predetermined detection location; an optical source configured to generate a plurality of laser pulses of at least one predetermined energy level and/or at least one predetermined wavelength, the optical source terminating at or near the probe tip surface, the laser pulses configured to cause generation of one or more transient vapor nanobubbles around malaria-specific nanoparticles at the predetermined location; and one or more acoustic detectors configured to detect acoustic pulses generated by the one or more transient vapor nanobubbles and output one or more signals indicative of the detected acoustic pulses to at least one processor, the one or more acoustic detectors being substantially flat and in close proximity with the optical source, wherein the optical source and the acoustic detector can be enclosed within the probe body. The optical source can comprise one or more optical fibers. The apparatus can comprise two or more optical fibers, wherein each of the two or more optical fibers can be located between two acoustic detectors The optical fiber can have a core diameter of about 50 μm to about 200 μm, or about 100 μm. The optical source can further comprise a laser pulse generator coupled to the one or more optical fibers. The acoustic detector can comprise a piezo element. The apparatus can comprise two or more piezo elements configured to detect signals of the same or different frequency spectra. The piezo element can comprise a navy type II or type VI material, or a composite material. The acoustic detector can comprise a substantially centrally located opening sized to accommodate the optical fiber. The acoustic detector can comprise a substantially flat disc, or two or more substantially flat discs or elements of other shape and acoustic properties. A tissue-facing surface of the acoustic detector can be about 0.1 mm to about 0.3 mm recessed from the probe tip surface. The sensor probe can further comprise a front layer between the probe tip surface and a tissue-facing surface of the acoustic detector. An outer wall of the optical fiber can be separated from a radially inner edge of the acoustic detector by about 0.01 mm to about 0.03 mm. An outer surface of the optical source can be separated from a radially outer edge of the acoustic detector by about 1.0 mm to about 1.5 mm. The sensor probe can further comprise a disposable cap. The laser pulses can be configured to cause generation of transient vapor nanobubbles around malaria-specific nanoparticles in blood and/or tissue. The predetermined detection location can be a patient's skin at the patient's wrist, ankle, lip, or tongue base or other locations. The predetermined detection location can be a surface of a flow cuvette with a flow path for a patient's blood or urine or other biological fluid sample. The probe tip surface can be configured to be covered with a layer of gel before being placed into contact with the predetermined detection location. The malaria-specific nanoparticles are located within the optical penetration depth beneath such location. The apparatus can further comprise a housing, wherein the probe body can be at least partially disposed within the housing; and a spring disposed between a proximal end of the housing and the proximal end of the probe body, the spring biasing the probe body toward a distal end of the housing.
An apparatus configured for diagnosing and/or treating malaria noninvasively can comprise a sensor portion, the sensor portion including: an optical source configured to generate laser pulses of at least one predetermined energy level, the optical source comprising an optical fiber terminating at or near a distal end of the sensor portion, the laser pulses configured to cause generation of one or more transient vapor nanobubbles around malaria-specific nanoparticles at the predetermined location; and one or more acoustic detectors configured to detect acoustic pulses generated by the one or more transient vapor nanobubbles and output one or more signals indicative of the detected acoustic pulses to at least one processor, the acoustic detector being substantially flat and in close proximity with the optical fiber at the distal end of the sensor portion; a housing, wherein the sensor portion can be at least partially disposed within the housing; and a spring disposed between a proximal end of the housing and the proximal end of the sensor portion, the spring biasing the sensor portion toward a distal end of the housing. The spring can be configured to be compressed when the apparatus is applied to a measurement site, the compressed spring forcing the distal end of the sensor portion into contact with the measurement site. The housing can comprise a patient interface at the distal end, the apparatus further comprising a liner substantially covering the patient interface when the apparatus is not in use. The patient interface can comprise an adhesive layer and/or gel layer.
A method of detecting malaria using any of the apparatuses disclosed herein can comprise instructing a laser pulse source to apply one or more laser pulses to a measurement site, any of the apparatuses disclosed herein being applied to the measurement site; receiving one or more signals from the acoustic detector of the apparatus, the one or more signals indicative of acoustic pulses detected by the acoustic detector upon the application of the one or more laser pulses to the measurement site; determining whether the measurement site is malaria-positive by: determining electronically a peak time of the one or more signals; comparing the peak time with a predetermined diagnostic threshold; and outputting a malaria-positive message if the peak time exceeds the predetermined diagnostic threshold, and outputting a malaria-negative message if the peak time does not exceed the predetermined diagnostic threshold. Determining whether the measurement site is malaria-positive can further comprise determining parameters from an amplitude, phase and/or shape of the signal. The method can also include using any of the apparatuses disclosed herein to scan a plurality of close locations to probe a sufficient volume of skin so as to improve detection of low level of malaria parasite density in the skin.
A method of detecting malaria using any of the apparatuses disclosed herein can comprise instructing a laser pulse generator to apply one or more laser pulses to a measurement site, the apparatus being applied to the measurement site; receiving one or more signals from the one or more acoustic detectors of the apparatus, the one or more signals indicative of acoustic pulses detected by the acoustic detector upon the application of the one or more laser pulses to the measurement site; and determining whether the measurement site is malaria-positive based on parameters from an amplitude, phase, shape, and/or a peak time delay of the one or more signals. Instructing can comprise instructing a laser pulse generator to apply one or more laser pulses of the same or different energy levels and/or wavelengths. Instructing can comprise instructing a laser pulse generator to route the laser pulses sequentially to a plurality of optical fibers. Receiving can comprise receiving a signal from a high-frequency one of the one or more acoustic detectors and a signal from a low-frequency one of the one or more acoustic detectors.
Various embodiments are depicted in the accompanying drawings for illustrative purposes and may not be drawn to scale, and should in no way be interpreted as limiting the scope of the embodiments. In addition, various features of different disclosed embodiments can be combined to form additional embodiments, which are part of this disclosure. In the drawings, similar elements have reference numerals with the same last two digits.
Although certain embodiments and examples are described below, this disclosure extends beyond the specifically disclosed embodiments and/or uses and obvious modifications and equivalents thereof. Thus, it is intended that the scope of this disclosure should not be limited by any particular embodiments described below.
The test parameters and experimental data provided in the present disclosure show preliminary results collected from in-field testing in early-stage human studies. They are disclosed herein as the evidence and examples of the first non-invasive detection of malaria in humans. They also provide insight into the principle of operation of laser-induced transient vapor nanobubbles for detecting malaria in human patients in non-invasively and/or minimally invasive manners. The sensor prototypes, experimental hardware and software are in early stages of development and the results obtained are thus process-dependent. The results and the processes used for collecting the results have not been scientifically peer-reviewed. However, the results were obtained under a controlled environment in blinded studies. That is, the personnel responsible for signal collection did not know the malaria status of patients at the time of signal collection. Further, human data were obtained and documented under an Institutional Review Board (TRB)-approved protocol and by using WHO-approved reference methods for determining the malaria status of each subject studied. Accordingly, it may be that the protocols, data acquisition and validation procedures, and scientific results and conclusions discussed herein could be subject to further analysis and future study.
The optical source can include a laser pulse generator 102 and one or more optical fibers 104. The laser pulse generator 102 can generate optical energy, which can be laser pulses of predetermined energy and/or fluence levels. The optical fiber(s) 104 can deliver the generated laser pulses to a location 26 on the test subject 20. Using the patient as an example, the optical fiber(s) 104 can direct laser pulses to any suitable locations on the patient, such as on the digits, hand, wrist, ankle (such as shown in
If the test subject 20 has been infected by malaria parasites, the test subject 20 can contain malaria-specific nanoparticles, such as HZ (hemozoin) nanocrystals. HZ (hemozoin) nanocrystals have a significantly higher optical absorbance than that of an uninfected red blood cell, uninfected hemoglobin, or major proteins in the red blood cell. The malaria-specific nanoparticles can likely be present at the location 26 as malaria parasites can travel to various locations in the patient by blood.
If the malaria-specific nanoparticles are located within a depth from a surface of the location 26 that can be penetrated by the laser pulses, laser-induced transient vapor nanobubbles (“nanobubbles”) can be generated at the location 26 underneath the surface of the location 26. Nanobubbles are a transient phenomenon. The generation of nanobubbles can produce sound waves.
The system 10 can have one or more acoustic detectors 106 configured for detecting the sound waves or an acoustic pulse of the nanobubbles generation. Close proximity of the acoustic detector(s) 106 and the acoustic pulse source, which is/are the nanobubble(s), can enhance the sensitivity and specificity of the acoustic detector(s) 106. This can be due to the acoustic pulse being stronger near the source than further away from the source, in particular for a point source. A nanobubble is a point source that generates spherical wavefronts. If the acoustic detector (s) 106 is(are) too far away from the source, the signal reaching a surface of the acoustic detector(s) 106 may be too weak to be detected. A commonly used acoustic detector can be a piezo element. The piezo element can generate an electrical charge in response to vibrations caused by the pressure wave.
In some embodiments of the present disclosure, the optical fiber(s) 104 and the acoustic detector(s) 106 are located in a single sensor probe 108. The single sensor probe configuration can be easier to use than having two separate probes for an optical fiber and an acoustic detector. The single probe configuration can also allow the acoustic detector(s) 106 to be closer to the nanobubble and to the optical fiber(s) 104 (which can reduce and/or minimize the angle of acoustic incidence) than if the optical fiber(s) 104 and the acoustic detector(s) 106 are in separate probes.
The system 10 can have one or more signal processors and/or controller 110 in electrical communication with the laser pulse generator 102 and/or the acoustic detector(s) 106. The one or more signal processors 110 can process the signals from the acoustic detector(s) 106 to determine if the signals are indicative of nanobubble(s) generation and thus for the presence of malaria-specific nanoparticles. In some embodiments, the signals from the acoustic detector(s) 106 can be amplified before being processed by the one or more signal processors 110. The one or more signal processors 110 can cause the processed signals and/or the detection or non-detection of nanobubble(s) generation to be displayed on a display device 112.
In some embodiments, the one or more processors 110 of system 10 can instruct the laser pulse generator 102 to emit a plurality of laser pulses at the location 26 to determine if there is nanobubble(s) generation at the location 26. As will be described in greater detail below, the laser pulses can have the same or different wavelengths and/or energy level. In some embodiments, the one or more processors 110 of the system 10 can instruct that the sensor probe 108 to expose to the laser pulse to different areas at the location 26. This can be achieved by mechanically scanning the surface with one optical fiber or by using multiple optical fibers (see
In some embodiments, such as illustrated in
The step 202 can include applying a layer of optically transparent ultrasound gel (or any other material to act as an optical and acoustic coupling media between the probe and the tissue) to a probe tip surface before applying the probe to the target location. Applying the probe to the target location can include pressing the probe tip surface and/or the layer of gel firmly into contact with a surface of the target location. The layer of gel can act as an optical and/or acoustic coupler by expelling air between the probe tip surface and the surface of the target location. In some embodiment, applying the probe to the target location can also include keeping a longitudinal axis of the probe generally perpendicular to the surface of the target location. The generally perpendicular probe can prevent air from entering between the probe tip surface and the surface of the target location, which can improve optical and/or acoustic coupling of the probe and the target location.
At step 204, the one or more signal processors can set an energy level of the laser pulse generator to a first predetermined level, E1. In some embodiments, E1 can be sufficient for generating nanobubbles around malaria-specific nanoparticles up to about 0.5 mm underneath a surface of the target location. In some embodiments, E1 can have an energy level of about 1 μJ to about 50 μJ, or about 10 μJ to about 15 μJ. In some embodiment, E1 can have a pulse rate of about 1 HZ (hemozoin) to about 100 HZ (hemozoin), or about 20 HZ (hemozoin) to about 50 HZ (hemozoin), or about 20 HZ (hemozoin), or about 50 HZ (hemozoin). The one or more signal processors can set more than one energy levels, such as two different energy levels. The one or more signal processors can also set one or more than one wavelengths for the laser pulses.
At step 206, the one or more signal processors can cause the laser pulse generator to apply one or more laser pulses having an energy level of E1. The number of pulses to be applied at a location can be predetermined, manually configured, and/or determined by the one or more processors based on certain algorithms. For example, the processors can stop additional pulses as soon as nanobubble generation has been detected, or continue instructing that additional pulses be applied until a predetermined number of pulses have been applied at the location. At decision block 208, the one or more signal processors can determine based on the signals outputted by the acoustic detector if one or more nanobubbles have been generated.
If the signal is not indicative of nanobubble generation, the one or more processors can optionally determine at decision block 210 if the pulse(s) applied at the step 206 include the last or final pulse to be applied to the patient for malaria detection. The number of pulses to be applied to each patient can be predetermined, manually configured, and/or determined by the one or more processors according to certain algorithms. The pulses can be applied to one or more measurement locations on the patient. The locations can be predetermined, manually selected by a user such as a clinician, and/or determined by the one or more processors according to certain algorithms. The one or more signal processors can instruct that the same or different numbers of pulses be applied to each location.
If the pulse(s) applied at the step 206 include the last or final pulse of the process 200, the one or more signal processors can output a message that no malaria parasite is detected at step 212. The message can be an audio signal, an optical signal, a text and/or symbol displayed on a display device, or a combination thereof. If the pulse(s) applied at step 206 do not include the last or final pulse of the process 200, the one or more processors can instruct that the probe be applied to another location at step 214. Pulse(s) can be applied to the new location to determine if nanobubble(s) generation can be detected at the new location. The energy level can be the same or different for each location.
If the signal is indicative of nanobubble generation, the one or more signal processor can also optionally determine at decision block 216 if the pulse(s) applied at the step 206 include the last or final pulse to be applied to the patient for malaria detection. If the pulse(s) applied at step 206 do not include the last or final pulse of the process 200, the one or more processors can instruct that the probe be applied to another location at step 218. Whether the signal is indicative of nanobubble generation can be determined by parameters derived from an amplitude, phase and/or shape of the signal.
If the pulse(s) applied at the step 206 include the last or final pulse of the process 200, the one or more signal processors can output a message that one or more malaria parasites are detected at step 220. In some embodiments, a signal indicative of nanobubble generation can include at least one (such as, two) spikes on a time-response trace (signal) received from the acoustic detector. Time taken for detecting the spike can be used to estimate a depth of the malaria-specific nanoparticles and/or malaria parasites. Time between the two spikes can characterize the maximal size of a detected vapor nanobubble.
At the step 206, if malaria-specific nanoparticles are present at the location, the laser pulse(s) applied to the location may also be sufficient for generating nanobubbles of a size that can cause mechanical damage to the malaria parasites. In some embodiments, the nanobubbles cause mechanical damage and/or destruction of the malaria parasites without harming uninfected blood cells and/or tissues.
If some individual signals obtained from healthy tissue look similar to nanobubble-specific signals associated with malaria disease and presence of HZ (HEMOZOIN) in the laser-exposed volume, groups of N signals (N ranges from 1 to 10,000), each in response to the corresponding laser pulse, can be analyzed statistically. Examples of statistical analyses can include using signal amplitude-derived diagnostic parameters, such as the normalized positive count, N, and the hemozoin index, HI, and a user-defined diagnostic threshold for the N and HI parameters. The normalized positive count, N, can be calculated using the formula N=Np/Nt, where Nt is the total number of the collected signals, Np is the number of signals with the peak-to-peak amplitude above a threshold T. The hemozoin index, HI, can be calculated using the formula HI=<A>−T*NNPCT, where <A> is an average peak-to-peak amplitude of the signals above T. Parameters above the diagnostic threshold would be indicative of malaria disease and parameters below the threshold would be indicative of healthy condition. In some embodiments, the hemozoin index of a malaria-positive signal can be about one order of magnitude greater than the hemozoin index of a malaria-negative signal. Examples of statistical analyses can also include a peak time-delay parameter and a user-defined diagnostic threshold for the time-delay. More details of the statistical analyses are described further below.
Additional diagnostic combinations in addition to applying one level of the laser pulse energy, one laser wavelength, and/or one type of the acoustic detector can be used to further improve the sensitivity and specificity of the detection of malaria-specific signal in the background of the bulk signal associated with healthy (malaria-negative) tissue.
In some embodiments, the process 200 can be repeated with a different laser wavelength than the wavelength used in the previous process 200. One of the two different wavelengths can be associated with a maximal optical absorption by malaria-specific nanoparticles.
In some embodiments, the process 200 can be repeated with a different laser pulse energy level than the laser pulse energy level used in the previous process 200. The difference in the nanobubble signals detected in the two processes 200 can be different due to the different number of bulk signals produced by the different energy levels.
The process 200 can be repeated with two different laser wavelengths and/or two different laser pulse energies, using the same sensor, or different optical fiber-acoustic detector combinations in the same sensor (which will be described below with reference to
The process 200 can also additionally and/or alternatively be performed with a sensor have two or more different acoustic detectors (described below with reference to
As the malaria diagnosis and/or treatment apparatuses and processes disclosed herein are based on photo-excitation of the malaria-specific nanoparticle, the process 200 can be reproducible and free from parasite resistance. As the generation of laser pulses and generation of nanobubbles take seconds, or at most several minutes, the treatment process 200 can be more efficient than traditional malaria diagnosis and/or treatment procedures.
Examples of the malaria diagnosis and/or treatment probe will now be described. As shown in
Before describing the sensor portion 310 in greater detail, a configuration of the malaria probe sensor portion 410 known in the art is described with reference to the schematic drawing in
The acoustic detector 430 can be generally a hemisphere with a substantially centrally located opening 436 to accommodate the optical fiber 420. The spherical acoustic detector 430 can circumferentially surround the optical fiber 420. In some instances, the spherical acoustic detector 430 can have a diameter D of about 4 mm. When a malaria-specific nanoparticles-generated nanobubble 22 (“nanobubble”) is generated under the surface of the target location 20, a distance d between the spherical acoustic detector 430 and the nanobubble 22 can be between about 2 mm to about 3 mm, or greater.
The spherical acoustic detector 430 can have some disadvantages. The pressure amplitude for the spherical pulse drops inversely proportional to the square of the distance from the source, as described above. As a result, the distance d can be large enough to result in substantial loss of the pressure between the nanobubble 22 and the acoustic detector 430 and hence in the reduced sensitivity of such sensor for detecting nanobubbles.
The acoustic detector 430 can be one or more flat piezo elements, which can be cheaper than spherical piezo elements. If the flat piezo elements are located in the same distance as the spherical piezo elements as shown in
Further challenges of using the spherical piezo element 430 can include the high cost of spherical piezo elements, and/or the complexity of incorporating the spherical piezo element 430 and the optical fiber 420 into the single probe. In some instances, a separate holder is required to hold the optical fiber 420 in the opening 436 of the spherical piezo element 430. The cost and complexity can make it difficult to mass-produce or commercialize a probe having a sensor portion 410.
The optical fiber 520 can extend substantially along a central longitudinal axis of the sensor portion 510. The optical fiber 520 can have a core diameter, or optical aperture, in the range of about 50 μm to about 200 μm, or about 80 μm to about 150 μm, or about 100 μm. The core diameter of the optical fiber 520 is small enough to reduce background bulk optical absorbance in order to maintain sufficient signal-to-noise ratio, and is not too small so it can easily miss the malaria parasite or malaria-specific nanoparticle.
The substantially flat acoustic detector 530 can form a disc with a substantially centrally located opening 536 to accommodate the optical fiber 520. The flat acoustic detector 530 can circumferentially surround the optical fiber 520. In some embodiments, the flat acoustic detector 530 can hold the optical fiber 520 in place without a separate holder. The flat acoustic detector 530 can be a piezo element. The piezo element can comprise a navy type II material, navy type VI material, any other piezo materials, or any combination of piezo and other materials.
As shown in
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Although the acoustic detectors 330, 530 are illustrated as a single flat element, the acoustic detector can also include two flat discs 530A, 530B, located on substantially opposite sides of the optical fiber 520, such as shown in
As shown in
Although a flat acoustic detector can be inferior, in theory, to a spherical detector for detecting the spherical pressure pulses from the nanobubble, the flat acoustic detector 530 is small in its size and in close proximity to the nanobubble such that the effect of the flat shape on the signal can be small. As a result, the flat acoustic detector 530 can improve the sensitivity and selectivity of malaria detection.
Moreover, the flat acoustic detector 530 does not have some of the above-described disadvantages associated with the spherical acoustic detector 430. When spherical pressure pulses arrive at the acoustic detector 530 having a small surface area and located in close proximity to the point source, the pressure pulses can arrive at the surface of the acoustic detector 530 at substantially the same or similar angles of incidence. As a result, it is less likely that opposite electrical charges are produced by the pressure pulses and the effect of de-phasing can be reduced and/or minimized. The flat acoustic detector 530 can thus improve the peak-to-peak amplitude of the spike. Larger peak-to-peak amplitude of the spike can improve the sensitivity and/or specificity of the sensor portion 510.
Incorporating the flat acoustic detector 530 and the optical fiber 520 into a single probe can be less complex than incorporating the spherical acoustic detector 430 and the optical fiber 420 into a single probe. As described above, the flat acoustic detector 530 can directly hold the optical fiber 520 without a separate holder. A flat acoustic detector, such as a flat piezo element, can also be significantly cheaper than a spherical acoustic detector, such as a spherical piezo element. As a result, the sensor portion 510 can be more suitable for mass production than the sensor portion 410.
With continued reference to
As shown in
In some embodiments, the sensor portion 510 can be reusable. A user can wipe the probe tip with alcohol to sterilize or disinfect the probe tip after each patient. In some embodiments, a disposable cap made of a thin film can be applied to the probe tip for each patient. The thin film can have little impact on the optical and acoustic contact between the sensor and the target location. The disposable cap can save a user's time for sterilizing or disinfecting the probe tip after each patient. In some embodiments, the film can have a thickness of about 1 μm to about 50 μm, or about 1 μm to about 20 m. The film may be covered with ultrasound gel or other biologically safe and optically- and acoustically-coupling material. The sensor probes described herein and a predetermined number of caps can also be provided in a kit. The number of caps can be sufficient for the life time and/or anticipated life time of the sensor probe in the kit.
The probe 1000 can have a sensor portion 1010 and a body portion 1005. The sensor portion 1010 can be coupled to the body portion 1005 and can extend distally from the body portion 1005. The sensor portion 1010 can have a smaller outer dimension than the body portion 1005. The probe 1000 can have a probe housing 1015 enclosing an optical fiber 1020 and a substantially flat acoustic detector 1030.
The optical fiber 1020 can extend substantially along a central longitudinal axis of the sensor portion 1010. The substantially acoustic detector 1030 can comprise two or more flat elements (such as discs) placed next to each other with the optical fiber 1020 running through a gap between the elements. As described above with reference to
The probe 1400 can have a sensor portion 1410 and a housing 1415. The sensor portion 1410 can include an optical source, such as an optical fiber (or more than one optical fiber) coupled to a laser pulse generator, and an acoustic detector (or more than one acoustic detector), such as a piezo element. The optical source can be configured to generate laser pulses of at least one (one, two, or more) predetermined energy levels and/or wavelengths. The optical fiber can terminate at or near the patient interface 1442. The laser pulses can be configured to generate one or more nanobubbles around malaria-specific nanoparticles that are under a surface of a measurement site. The measurement site can be on a patient's body, or a flow cuvette described below. The acoustic detector can be configured to detect acoustic pulses generated by the one or more nanobubbles and output a signal indicative of the detected acoustic pulses to at least one signal processor. The acoustic detector can be substantially flat and in close proximity with the optical source.
The sensor portion 1410 can be located at least partially within the housing 1415. The housing 1415 can include a distal end 1440 and a proximal end 1444. At the distal end 1440, the housing 1415 can include a mounting component 1442. The mounting component 1442 can provide a greater footprint of the probe 1400 on the measurement site, such as the patient's skin, than the contact between the sensor portion 1410 and the patient's skin. In the illustrated embodiment, the mounting component 1442 can be generally ring-shaped to accommodate the sensor portion 1410 located generally at the center of the patient interface 1442.
The mounting component 1442 can include a patient attachment mechanism, which can have an adhesive layer, a gel layer, and/or otherwise. The mounting component 1442 can also include an adhesive layer and/or gel layer that is covered by a liner 1441 when the probe 1400 is not in use. When in use, such as shown in
The probe 1400 can be coupled electrically to the laser pulse generator, the signal processor, and/or the display that are disclosed herein. The housing 1415 of the probe 1400 can have an opening 1445 (shown in
As shown in
As shown in
Embodiments of the malaria probe disclosed herein can be used in various applications to diagnose malaria. For example, a malaria probe, such as one having the sensor portion 510 of
As shown in
The transdermal (skin) application can be effective not only in detecting and/or killing malaria parasites in the patient's blood. The malaria probe disclosed herein can have sensitivity and/or specificity to detect tissue-sequestered malaria parasites and/or hemozoin nanoparticles by generating nanobubbles around the malaria-specific nanoparticles in the micro-capillaries in the tissue.
The malaria probe disclosed herein not only has in vivo applications, but can also be used to detect malaria parasites ex vivo, to analyze blood, urine or other body fluids.
The application of the probe on the liquid sample (for example, such as the peripheral blood) and on the skin stem from different biological mechanisms: active disease (peripheral blood) vs transmission (skin). Hence the peripheral blood cannot be analyzed “through the skin” and parasites or hemozoin properties do not correlate in peripheral blood and skin. Also, these applications have opposite scientific back-ups: well-established for blood and thus may provide an additional test if conventional blood test described above for malaria turns out to be inconclusive, and almost non-existent science for malaria in skin.
It is the skin rather than the blood that may be more challenging in the eradication of malaria. Malaria transmission can start with mosquitoes picking up parasites from the skin of an infected patient, specifically, the gametocytes from the subcutaneous layers of skin of the infected patient. Among all blood stages of parasites, these are the gametocytes that can deliver the maximal nanobubble signal because they have the highest quantity and size of hemozoin particles. The gametocytes, after becoming biologically inert, may last for many months in the skin. A transmission of malaria requires gametocytes be available for mosquito bites, that is, in the subcutaneous upper skin layer, which can be less than 1 mm in thickness. This biological mechanism delivers hemozoin to where it can meet the laser pulses emitted from the malaria probe examples disclosed herein (for example, at about 0.1 mm to about 0.5 mm optical penetration depth). The higher level of hemozoin in gametocytes, compared to other stages of parasites, can result in the higher nanobubble signal as found both in the laboratory and human studies disclosed herein.
The systems for noninvasively diagnosing and/or treating malaria can have multiple applications. The systems can have a diagnostic application for determining if a patient carries malaria-specific nanoparticles, hemozoin. If nanobubble(s) generation is detected using the processes, parameters and thresholds described herein, the patient can be diagnosed as malaria positive, including the residual malaria without active disease symptoms. If the nanobubble(s) generation is not detected, the patient can be diagnosed as malaria negative. The malaria-positive patients may have been treated when the nanobubble generations have caused mechanical destruction of the malaria parasites. The malaria-positive patients may additionally or alternatively be treated with anti-malarial drugs.
The systems can also have an epidemiological application for determining a malaria-infected region and/or the foci of malaria transmission. Caregivers typically provide anti-malarial drugs to a large population when there is an outbreak of malaria in a region. Regulators such as the WHO also issue rules and reports on the administration of anti-malaria drugs to various populations. As current malaria detection methods have difficulty detecting asymptomatic, sub-potent, past malaria infections, especially when peripheral blood is malaria-free, and are thus limited in locating where malaria originated, providing treatments to a large population, such as across an entire country, may be the only way to prevent transmission of malaria within that population. Anti-malarial drugs can have undesirable side effects, such as causing miscarriage in pregnant women, heart failures and other dangerous conditions. It is therefore desirable to narrow down the malaria-infected region/populations as much as possible to avoid administering anti-malarial drugs to people who may not need the drugs and/or may be harmed by the drugs.
The systems and processes described herein provide ways to narrow down the malaria-infected region. The human body carries malaria-specific nanoparticles, such as the HZ (hemozoin) nanocrystals, so long as the body was once a host of active malaria parasites. Even when the body no longer carries active malaria parasites, for example, if the malaria parasites went dormant and/or if the body had been infected by malaria parasites in the past, the HZ (hemozoin) nanocrystals can still be present in the body, especially in the skin. The ability of the system to detect the HZ (hemozoin) nanocrystals with or without an active malaria parasite using laser-induced nanobubbles can allow detection and/or narrowing down of geographical regions and population where malaria is endemic, and/or where malaria originated. Caregivers can treat a smaller population in the narrowed-down region, such as a small village, with anti-malarial drugs or other malaria treatments in order to contain the spread of malaria. The more localized and/or targeted administration of anti-malarial drugs can be effective in malaria eradication without unnecessarily dosing a large population with the drugs.
For geographical mapping of the malaria transmission through the mass screening of the population, the confirmed geographical location of human subjects can be mapped in a way such that the mapping shows the relative level of malaria positive (hemozoin-positive) signals for specific areas where the screening (collection of the signals) is performed. This can be achieved by calculating the relative level of malaria- or hemozoin-positive subject normalized by the total number of screened subject per specific area (for example, a village, a border checkpoint, a clinic, or other specific geographical location). Such an approach would be more efficient for a settled “stationary” population. The mapping of dynamic “transit” population (such as at a border checkpoint or an airport) would require additional information on the geographic origin of human subject. In either case, the absolute number of positive detections can be considered also after being normalized by the total number of subjects screened from the area in question.
The mapping of the malaria transmission may additionally involve the analysis of local demographics so the signals are analyzed not just as a function of the coordinates of the screened area but also of the demographic, climate and medical parameters of the screened area (age, gender, ethnicity, income, presence of other diseases, time of the year, stage of malaria transmission season, etc.) and other factors which are related to the transmission of malaria. Such analysis can be performed locally or remotely by uploading the signals and other area-specific data to the remote server. Such analysis would result in an epidemiological “maps” of malaria transmission which may be very useful for the treatment, elimination and prevention of malaria. As shown in
Collection and analysis of malaria population data will now be described in more detail. It is often reported that there is insufficient information with which to deploy the available resources for controlling the transmission malaria. Many malaria-endemic countries still suffer from weak health-management information systems and often lack vital registration. At a global level, only around 10% of estimated malaria cases are detected. Many patients with suspected infections receive empiric antimicrobial therapy rather than appropriate therapy dictated by the rapid identification of the infectious agent. The result is overuse of a small inventory of effective antimicrobials, whose numbers continue to dwindle due to increasing levels of antimicrobial resistance. As shown in
In geo-tagging of malaria, primary data can be further combined with fingerprinting device incorporated with the malaria sensors for reliable identification of all screened subjects. With a properly designed and connected global data base, such information may be used by border and passport control authorities to track the malaria carriers and thus to contain the malaria infection and prevent its spread.
The table below describes the clinical and mass-screening applications of the malaria diagnostics disclosed herein.
As described above, in addition and/or alternative to monitoring the parameters derived from the amplitude of acoustic signal when one or more predetermined laser pulses are applied to a measurement site, the systems described herein can independently detect nanobubbles generated around malaria-specific nanoparticles in a tissue based at least in part on a signal peak time-delay. The signal peak time-delay can be used as an additional diagnostic metric, which is independent of the metrics derived from the signal amplitude, such as the normalized positive count, N, and the hemozoin index, HI described above. The hemozoin index can represent a relative signal amplitude above an amplitude threshold. The normalized positive count can represent relative number of signals above the amplitude threshold. The peak timing metric can be used together with the signal amplitude metrics (N and HI) to increase the diagnostic sensitivity and specificity of the clinical diagnostics and/or mass screening.
When the tissue is infected with malaria, the source of the acoustic pulses also includes nanobubbles generated around malaria-specific nanoparticles. The acoustic signal from nanobubbles can be different from the background acoustic signal from the skin. The acoustic signals from nanobubbles can be delayed by the time determined by the depth of nanobubble and the speed of sound.
Tissues with subcutaneous malaria-specific nanoparticles result in the maximal nanobubble-emitted pressure pulse, which is at some depth from the skin surface. The distance traveled by the pressure pulse to the sensor and from subcutaneous parasites, including the malaria-specific nanoparticles, can be higher (such as slightly higher) than the distance a thermos-elastic wave travels from the skin surface. The distance traveled by the pressure pulse from nanobubbles can be determined by the depth of the malaria-specific nanoparticles, such as HZ (hemozoin) nanocrystals. The malaria-specific nanoparticles can be located at the depth from about 10 μm to about 400 μm, or to about 500 μm below the skin surface. About 500 μm below the skin surface is not the location limit of the malaria-specific nanoparticles, but can be the maximal depth of the optical penetration of the laser pulse which still can generate a nanobubble around malaria-specific nanoparticles. Due to the additional travel distance, the acoustic pulses from the nanobubble generation also arrive at the sensor probe with a time delay compared to the background acoustic pulses from the skin surface. The time-delay due to the additional travel by the acoustic pulses can be determined by the speed of sound in tissue and the depth of HZ (HEMOZOIN) location. The time-delay can be about from 40 ns to about 300 ns, or to about 333 ns (assuming that sound travels at 1600-1800 m/s in the tissues).
When comparing the acoustic signals from malaria-negative (healthy) tissues and malaria-positive tissues, such as shown in
The depth of the malaria-specific nanoparticle can be calculated by multiplying the speed of sound in the tissue and the time delay. As shown in
The time-delay in the malaria-positive signal and the background signal can be detected with an ultrasound detector with sufficient temporal resolution, such as the acoustic detector described herein. The ultrasonic detector can have a frequency of at least about 4 MHZ (hemozoin) or more to detect a signal peak time from the malaria-positive and malaria-negative signals.
A peak timing-delay diagnostic threshold can be used as an additional and/or independent malaria diagnostic criterion. Signals with the peak timing below the diagnostic threshold can be assumed to indicate a malaria-negative status, and signals with the peak timing above the diagnostic threshold can be assumed to indicate a malaria-positive status. The diagnostic threshold can be predetermined, for example, using empirical data. As will be described below, the threshold can be determined based on the peak timing-derived time histograms.
At step 1508, the one or more signal processors can receive an acoustic signal from the acoustic detector of the probe. The signal can be indicative of the background acoustic pulses generated by the skin, and/or the acoustic pulses from the generation of nanobubbles (if malaria-specific nanoparticles are present at the target location). At step 1510, the one or more signal processors can determine a peak time of the signal. At decision block 1512, the one or more signal processors can determine whether the peak time of the signal exceeds a predetermined diagnostic threshold. The diagnostic threshold can vary depending on the type of sensor, the type of target location or measurement site, the depth of the malaria-specific nanoparticles, and/or the species of the malaria parasite.
If the peak time does not exceed the diagnostic threshold, the one or more signal processors can output a message that no malaria is detected at step 1514. If the peak time exceeds the diagnostic threshold, the one or more signal processors can output a message that malaria is detected. At decision block 1512, the one or more signal processors can also combine the determination based on the peak time with the signal amplitude parameters, such as the N and HI values. For example, if the signal peak time does not exceed the diagnostic threshold, but the signal amplitude parameters are highly indicative of a malaria-positive status, the one or more signal processors can output a malaria-positive status message. At the decision block 1512, the one or more signal processors can also compare the signal amplitude parameters with a signal amplitude parameter threshold instead of comparing the peak time of the signal with the peak time threshold.
At step 1518, the one or more signal processors can also optionally determine the depth of the malaria-specific nanoparticle based at least in part on the peak time of the malaria-positive signal.
As shown in
Table 1 below illustrates an example two-sample t-test performed on the results of the human study of the application illustrated in
As shown in the table, there can be a 110/130 ns average/median delay in the timing of peaks of malaria-positive signals compared to malaria-negative signals. The mean time-delays can be statistically significant, regardless of whether equal variance is assumed.
The human subjects were also tested for malaria using standard microscopy and PCR tests of peripheral blood samples. As shown in
As also shown in
In another example study of nanobubble based malaria detection, different types of sensor probes were used on the patient's ankle, back of hand, blood, and urine samples respectively and different diagnostic parameters were used. Data was collected from clinical studies using the malaria sensor described herein in various stages.
In addition to peak time as the diagnostic parameter, the signal amplitude-derived diagnostic parameters were used for some of the tests on the ankle. The subjects were tested for malaria using a signal amplitude-based diagnostic parameter (N-HI) for the rest of the tests.
The subjects were also tested for malaria using current diagnostic tools such as the RDT, PCR and microscopy. In
Table 2 below summarizes the sensitivity and accuracy (as defined above) of the various tests in the example study.
Using the sensor probe such as shown in
In addition to the skin tests, blood and urine tests were performed. 3 flow tests were performed with 1000 signals each. Capillary (peripheral) blood was taken with a finger prick. Reference methods, microscopy using the peripheral blood, RDT, and PCR, were also performed using peripheral (blood) samples.
The signal analysis can use the following equations for calculating the signal amplitude metrics in the liquid (urine and blood) sample and non-invasive skin tests. The normalized positive count Nnorm can be calculated as
where Npos is the number of signals above a threshold, and Ntotal is the actual number of laser pulses. The normalized amplitude above the amplitude threshold, HI, can be calculated as
where T is the threshold signal amplitude and A is the peak-to-peak amplitude for the signals equal to or above T.
This diagnostic procedure was found to be safe and without any detectable damage to human skin.
Table 3 below summarizes the sensitivity and accuracy (as defined above) of the various test in the example study. As shown, the worst signals (lowest amplitude, minimal separation of negative and positive components) were observed in the studies for an inner lip and a tongue base.
Data was collected in two studies from subjects in Sumatra and The Gambia. In Sumatra, the collected data was related primarily to the malaria species P. Vivax. In The Gambia, the collected data was related primarily to the malaria species P. Falciparum. Each study included two stages: a clinical application stage (Stage 1) and a mass screening stage (Stage 2). The clinical stage was further divided into Stage 1A and 1B. Additional details of the studies are provided in Table 4 below.
As determined in the validation stage (Stage 1B, blinded) and shown in Table 5 below, when the malaria status of the blood and skin correlate, such as when malaria parasite and/or hemozoin are present in both the blood and in the skin, the sensitivity and specificity of the method of malaria detection using the sensors disclosed herein can be high. Throughout this disclosure, “healthy” and “negative” both denote subjects without malaria infection.
P. Falciparum
P. Vivax (Sumatra)
For both types of malaria, in the above shown two independent blinded studies, a good separation of data for positive and healthy subjects for suspected clinical cases were found. As shown in
As determined in the mas screening stage (Stage 2) and shown in Table 6 below, when the malaria status of the skin and the blood differ, such as when there is no malaria parasite and/or hemozoin in the blood but sequestered malaria parasite and/or hemozoin in the skin, the method of malaria detection using the sensors disclosed herein cannot be benchmarked against the standard methods on peripheral blood. Another skin-based reference method is required to validate the method using the sensors disclosed herein.
P. Falciparum
P. Vivax (Sumatra)
In the study of both clinical and asymptomatic cases, the malaria status was also determined through the microscopy and PCR analyses of peripheral blood samples. For clinical cases that are associated with the acute disease, which in turn develops in the peripheral blood, there was a good correlation between the blood malaria status and hemozoin-generated vapor nanobubble data as found both for Plasmodium Vivax and Plasmodium Falciparum types of malaria parasites. As determined by comparing the data from The Gambia in the clinical application stage (Stage 1, see
As described above, the biological mechanisms causing the difference in the group average values could be at least the sequestration of parasites and hemozoin from the peripheral blood into subcutaneous microvasculature. As shown in Table 8 below, it can be typical for patients having late-stage malaria parasite infections, which would have produced a high level of hemozoin, to have more hemozoin accumulated in the skin than in the blood, or even to have malaria-free peripheral blood but still have parasites and/or hemozoin in their skin. In skin, the sequestered parasites and/or hemozoin can persist for months, whereas the parasites and/or hemozoin can clear from the peripheral blood in days. Sequestered parasites and/or hemozoin can be responsible for lethal complications in the clinic, such as uncontrollable transmission and relapses. Sequestered parasites and/or hemozoin can represent a hidden source of malaria injections. As described above, no standard method based on blood testing can detect the tissue-sequestered malaria parasites and/or hemozoin. Skin can also be a better source for malaria screening than standard methods using peripheral blood.
The differences observed above indicate that many local subjects from endemic area with malaria-negative blood still had skin which delivered hemozoin-generated vapor nanobubble-positive signals and hence potentially had parasites/hemozoin not found in their peripheral blood. The current malaria science suggests that there is no correlation between the malaria status of the peripheral blood and malaria transmission (which is related to the skin level of parasites, especially, gametocytes). Therefore, the asymptomatic data may support the hypothesis about high level of sequestered parasites and hemozoin in skin. Such cases may not represent clinical concerns, but they may represent transmission concerns even though such human subjects are not detected as malaria-positive with current standard malaria tests. That standard PCR analysis of most of such hemozoin-generated vapor nanobubble-positive subjects came out as negative. The non-invasive skin data suggest a possibility to use the hemozoin-generated vapor nanobubble method for the detection (and screening) of malaria transmission status of human subjects through a rapid and non-invasive hemozoin-generated vapor nanobubble test. The clinical studies suggest that a skin may be a better source for malaria detection and screening compared to the current standard, a peripheral blood, for the screening of malaria transmission. As shown in
As described above, examples of the malaria sensor disclosed herein can deliver laser pulses into the skin of a test subject (such as a patient) through an optical fiber. In some embodiments, the optical fiber can be a multi-mode optical fiber. The sensor can collect a spherical pressure pulse from a subcutaneous source, which can be the generation of a nanobubble around a malaria-specific nanoparticle, such as a hemozoin nanoparticle. The sensor can also amplify the electrical signal indicative of the pressure pulse to a level that can be analyzed.
The nanobubble-based malaria detection mechanism is different than the photoacoustic mechanism described above with reference to
Signals from experimental models and human subjects described below show that the transient vapor nanobubbles were directly detected in the skin during a non-invasive test and that the nanobubble signals correlate to the malaria-positive status.
In an experiment using gold nanofim as a source of vapor nanobubbles as a reference model, such as shown in
From the bottom of the glass slide, a single short 532 nm laser pulse was focused onto the gold film through the glass. This resulted in the generation of semi-spherical transient vapor nanobubbles in water and near the gold/glass surface. The maximal size (hence the lifetime) of a nanobubble was controlled through the energy of the laser pulse (through the amplification settings in a laser remote control). In this model, a vapor nanobubble acts as a point source which generates a pressure pulse with spherical wave front.
A calibrated reference hydrophone HNC 1000 or HNC 0400 (which may be the default choice) may be mounted, for example, at a slight angle above the gold surface. The hydrophone has a round element with the diameter 0.4 mm and the spectrum shown in
As shown in
A ratio of the size of the piezo element in the hydrophone to the hydrophone-to-target distance can characterize a detection regime of the hydrophone as a far field (<1) and near field (≥1). In the far field, the sensor detects almost a flat wave so the detection conditions (angle of incidence and phase of the pressure pulse front) remain substantially equal across the whole surface of the sensor. In the near field, the sensor detects a spherical wave and the detection conditions significantly vary across the sensor surface. Further, any additional lateral shift of the sensor off the center axis (a factor that is unavoidable when the optical fiber is placed in the center and the piezo element has some lateral shift by definition) adds to the effective size of the sensor geometry. In the far field in
In the near field, the nanobubble signal becomes distorted due to several reasons: (1) the angle of acoustic incidence becomes too high, which reduces the sensor sensitivity and the signal does not increase despite shorter distance to the target; (2) the piezo element becomes too large in size, causing significant dephasing (mismatch) in the pressure-to-charge conversion, which broadens the signal spike and further reduces its amplitude. The lateral shift of the piezo element relative to the pulse source (a feature generally unavoidable for the malaria sensor with the optical fiber in the center, as described above) causes further broadening of the signal spikes and the decrease in the amplitude of the signal spike. Those effects can be explained by the increasing angle of acoustic incidence and the resulting dephasing of the piezo-effect in a piezo element, especially if its size is relatively large compared to the sensor-to-target distance. Further, significant lateral shift reduces the difference between the nanobubble-specific and bulk background signals (which will be described in greater detail below), making it harder to differentiate those two signals.
In
Three testing models can be constructed using the optical fiber-hydrophone arrangement illustrated in
For the water model of vapor nanobubbles, the transient vapor nanobubbles in water can produce a two-spike signal with the corrected spike amplitude in 2-3 mV range (with a 10-fold amplification of the hydrophone) for a nanobubble of 1 microsecond lifetime, which corresponds to the maximal diameter of 10-20 um approximately. The nanobubble expansion and collapse are nearly symmetrical, the second spike generated by the collapse can be close in amplitude to the first spike caused by expansion and such nanobubbles create no tensile stress in water.
A nanobubble in skin can be modeled with a human skin sample surrounding the nanobubble generated by the gold-water model described above. The test sample included a gold nano-film deposited on the microscope slide glass and the human skin sample on top of the gold film, in physiological solution or in water. The skin sample was dark human skin, 250 um thick, and including epidermis and dermis. A drop of water was deposited on the sample to couple the sample to the hydrophone. In this model, a nanobubble formed by optical excitation pulse as described above with reference to
After a sample of dark human skin of 250 um thickness was placed on top of the glass, the energy deposition and vaporization processes have not changed because the laser-target interaction and target properties have not changed. However, as shown in
The nanobubble energy is dissipated during the expansion stage in the skin model. As shown in
The skin also has significantly influenced the collapse of a nanobubble (the second spike), sometimes up to its full damping sometimes as shown in
Table 9 below includes statistics for signals in skin and water under identical laser excitation and acoustic detection.
49 ± 0.19
It is possible to expect irreversible local structural changes in the exposed skin after each nanobubble. A small air bubble may remain in skin for up to 30 seconds. Such changes may cause (1) delivery of new parasites into the exposed volume after the local implosion of the skin when the void created by a nanobubble finally collapses, and (2) a change of the local optical scattering properties of the skin (so the detection of “after-bubble” may be an option detection of a parasite).
It is also possible that at a smaller scale of hemozoin-generated vapor nanobubbles (the maximal diameter 10 um or less), the plastic deformation and associated damping of the nanobubble collapse may be the minimal and thus the quality of the second spikes may improve. However, such second spikes may be located close in time to the background spike (<300 ns interval), and may have a small amplitude. Such second spikes may require more precise detection.
Accordingly, in skin (unlike water), a more reliable signal for a transient nanobubble may be the first spike associated with its expansion. The second (collapse) spike has strongly damped behavior and appears to be unstable due to the plastic deformation of skin and viscous damping. Further, the simultaneous generation of several closely located nanobubbles of different maximal size (and hence lifetime) may amplify the first spike but may broaden/diffuse the signal of the second spikes (since individual nanobubbles collapse in different time).
Acoustic detection was performed using a HNC 0400 (0.4 mm) reference hydrophone (
As shown in
As shown in
The following metrics were observed for the signals in the model shown in
For the second spike, the time interval (lifetime) observed was in the range of 0.8-2.8 us. The second spikes were not observed below 0.7 us, maybe due to the ripple in the baseline. This spike was caused by the collapse of a vapor nanobubble and hence was determined by the mechanical properties of the skin. The skin influence on the second spike in parasite-treated skin appeared to be very similar to that observed in the gold-skin model. Several typical features were observed. In the initial time position, the amplitude of the signal decreases to 0% (the spike disappeared) when the same location is exposed to the second or next laser pulses. Additional spikes were observed at shorter time intervals (the “move-in” effect), which was associated with the generation of smaller vapor nanobubbles as consecutive laser pulses were applied to the same skin location. To quantify parasite-specific features, eight metrics were introduced:
These metrics were analyzed for intact and parasite-treated dark skin of 250 um thickness, all in response to 671 nm 15 uJ laser pulse delivered via the optical fiber with a 105 um core. Table 10 illustrates a comparison of intact and parasite-treated skin samples, dark, 250 um thick.
The detailed analysis of the first spike (background signal and the effect of the expansion of a nanobubble) is provided herein. Regardless the shape of the peak (a single or dual peak), its amplitude in response to the first laser pulse and the decay were different in intact and parasite-treated skin (
The detailed analysis of the second spike (the effect of a nanobubble collapse) is provided herein. In
The main features of
In summary, signals obtained from a human skin sample with residual parasites had shape and metrics similar to those of the gold-skin nanobubble model. This similarity appears to validate the signals detected in a parasite-treated skin as signals of vapor nanobubbles. The properties of two main nanobubble signal components, the first and second spikes in the human skin sample are summarized in Table 11A.
As shown in Table 11A, vapor nanobubbles are generated in skin around Plasmodium Falciparum human malaria parasites in the human dark skin. Plastic and viscous properties of the skin can dampen the collapse stage of the nanobubble, the effect resulting in an unstable second spike associated with the dampened collapse. The acoustic detection of parasite-generated nanobubbles disclosed herein may be more effective for relatively large nanobubbles with the lifetime above 0.8 us, which corresponds to the maximal diameter of a nanobubble about 10 um. The background signal due to optoacoustic emission by melanin can be high and create one of major challenges in the generation and detection of nanobubbles around malaria parasites in a dark skin.
Several options are available for improving the nanobubble method disclosed herein. It may be possible to modify optical excitation in the way its fluence at the melanin depth is reduced (for example, via side launch of the focused launch of a pump laser pulse as shown in
The propagation of the excitation laser beam and its absorption in melanin-rich dark human skin was modeled with a computer program. The skin was modeled through 7 layers to describe stratum cornea, epidermis with the layer of melanin and upper layers of dermis. Optical scattering of the excitation beam by each skin layer and its optical absorption were analyzed for specific wavelength of the excitation laser pulse. The program and the model were used to compare the propagation of the excitation laser beam in water and in human dark skin (
Next, the monte-Carlo simulation method has been applied to analyze the propagation of individual photons through the skin under direct delivery from the optical fiber and the focused deliver from the top and from the side (see Table 11B below).
As can be seen from the results of the computational modeling, the background signal amplitude-driving laser fluence at the level of melanin in skin decreases in both cases of the focused beam compared to that for the standard launch with an optical fiber. Further, the fluence of the excitation beam at the level of possible location of malaria parasites increases (compared to that for the standard launch with an optical fiber) in both cases of the focused launch. This computational model has been used to design the experimental model with dark human skin and malaria parasites in the skin, and to compare the standard launch of the excitation laser beam and the side-focused launch.
The side launch of the excitation laser pulse spatially decouples the source of the background signal (melanin in human dark skin) from the source of malaria signal by shifting the source of the background pressure pulse out of the optimal detection angle of acceptance of the acoustic detector (
The goal of the delivery of the pump beam is to minimize the optical fluence at and associated thermal impact of the background skin (especially, upper level-located melanin) and to maximize the optical fluence at the depth of parasites. This is achieved by focusing the probe beam and by launching it as some angle so the background skin volume is spatially decoupled from the skin volume with hemozoin and or parasites.
Such spatial decoupling of the target (hemozoin) and background (melanin) was validated in dark human skin (rich with melanin) and human parasites placed at the depth of 250-300 um below the skin surface. Ultrasonic signals were compared for the background and hemozoin-generated vapor nanobubble to those obtained under the regular optical delivery through the flat 105 um core optical fiber.
Compared to a standard or direct optical fiber launch in intact dark skin (
Additional improvement of the optical delivery was achieved by suppression so called “hot spots” in the pump laser beam (which cause additional false-positive signals generated by melanin). Such suppression was achieved by homogenizing the laser beam intensity inside multi-mode optical fiber by increasing its lens from 1-2 μm to 12 m (see Table 11D).
A comparison of the skin model and the field human data will be described below.
The propagation of acoustic pulse from the VNB through the skin to the acoustic detector located at the skin surface is modeled. For the focused beam of 300 um diameter at the skin surface, a propagation and detection of a single acoustic pulse emitted was estimated, for example, during the expansion of a vapor nanobubble around malaria parasite as shown in
Optical excitation used the same laser pulses as shown with reference to
The malaria sensor probe can include a low-speed sensor with a relatively poorer temporal resolution and poorer acoustic damping, compared to those of the 0.4 mm hydrophone). Co-registration was achieved with the 12 MHZ (hemozoin) reference hydrophone (1.0 mm element, Onda HNC1000) on top of the sample. Due to acoustic reflections from a hard surface of the malaria probe, the reference hydrophone detected both direct signals and their echoes. The only echo-less signals were those originated from the surface of the malaria probe. For the malaria probe, the detection conditions were similar to those in human field studies.
Validation of nanobubble detection with the malaria probe (such as shown in
The sensor signals in the human skin model were obtained without and after adding parasites to 250 um thick dark skin (
Statistical analysis of the malaria sensor and hydrophone signals is described below. There was an increase in the peak-to-peak amplitude of the first spike and the time-shift in the signals from the parasite-treated skin. As shown in
Statistical analysis of the time-shift and amplitude of the first spike as described with reference to
The corresponding hydrophone data revealed a similar time-shift trend (Table 13). Average values for the first spike position and interval for three independent experiments with black skin sample of 200-300 um thickness (every day a new sample was prepared, and the skin thickness variability may exceed 50 um) were calculated. The number of signals in each group was 16-20.
The observed influence of parasites on the first spike was further analyzed by using HI-N signal amplitude metrics and by varying the time window where they were calculated for. In the full time range (similar to how it has been done in human studies, with the similar amplitude threshold, see Table 13), adding parasites has increased values of both N and HI by 2.5-14 fold. The values of N and HI were close to those obtained above for the analysis of the first spike only. This may indicate a visual fact that, in the full time range mode, the first spike dominates the signal. Further, in the time range from 2.0 us (where nanobubble signals were observed for the same sensor in the experimental water model of nanobubbles and where nanobubble-like signals were observed in the non-invasive human study in The Gambia) to 2.6 us (the max time range to avoid any echo signals), a 2-6 fold increase in both metrics was observed after parasites were added.
The time-shift observed for the first spike after parasites were added matches the hydrophone data for the same samples. A similar direction and value of the time shift was observed with the hydrophone for the first spike (note an opposite location of the hydrophone hence an opposite time-shift). However, the malaria sensor may not resolve melanin and parasite spikes as the hydrophone did. Instead, it appears that sensor has integrated closely located spikes into one spike with varying amplitude as adding parasites has increased the peak-to-peak amplitude of the first spike detected with the sensor but did not increase the amplitude of any spike in the hydrophone signal. These differences in signal parameters could be caused by a narrow acoustic frequency bandwidth of the sensor compared to that of the hydrophone. Nevertheless, the optimization of the time window range and the amplitude threshold allows for distinguishing intact and parasite skin even with a slow-speed sensor (see, e.g.,
In the field human studies the malaria sensor returned typical signals for healthy (n=25) (
Accordingly, new diagnostic metrics that can account for the second spike correlating to the collapse of the nanobubble may be needed.
The HI-N amplitudes in
The N-HI diagram (for all subjects, single data point corresponds to a single subject) shows the following statistical properties of signals. The metrics were calculated for the similar settings for the time window to include both the first spike and secondary spikes and an amplitude threshold of 0.16 V. Table 15 shows group-averaged values (N=30 for malaria, N=25 for healthy). For human field data, one specific location, an ankle, was analyzed. When assuming a time-shift to the right of the first spike and setting the time-window at 1.02 us to 5.0 us, the amplitude threshold unchanged Tha=0.16 V, the separation of healthy and malaria data improves, their group averages increasing by more than one order of magnitude and two groups being statistically significantly different (p<0.001). Metrics of the first spike, with the time window optimized for the time-shift observed, provided the best diagnostic separation for signals obtained with the slow-speed sensor.
When applied to the time window associated with the second spike of a nanobubble collapse, at time window 2.0-5.0 us, only peak-to-peak amplitudes (without subtracting a non-flat baseline) were analyzed. This limitation of the analysis decreased the separation between healthy and malaria-positives but still indicated some statistically significant difference between these subject groups. The distribution of HI-N data in the model (
In the skin model experiment, the incidence of the second spike was lower than in field human data. This can be explained by two factors: (1) mechanical properties of live (humans) skin are more favorable for the collapse of nanobubble compared to properties of dead skin (the model), and (2) real human skin had more parasites and they also were closer to the surface compared to the condition of residual single parasites at 250 um depth in the model.
Table 16 below summarizes the group-averaged N nb values and additional details of
Several factors influenced the occurrence of the second spike: (1) not all nanobubbles collapsed, not all collapsed nanobubbles were detected, only those with the lifetime above 1 us were detected, because the second spikes of smaller nanobubbles were obscured by a non-flat baseline of the signal output of malaria sensor. The occurrence of collapse spikes (2nd spikes) was random through the test run from 1st to 60st signals. They were not linked to specific, for example, initial laser pulses like they were in the skin model where a strong signal decay was observed. This difference between the dead and live skin suggests that the laser-exposed volume in live skin does not remain static during the test (which can be about 3 seconds) and new hemozoin targets can enter the volume during the test. This may be the result of plastic deformation of skin or cracks induced by nanobubbles and the resulting “mixing” of the skin volume.
In summary, in several independent experiments, adding parasites (including the residual levels of parasites) to the bottom of the human dark skin sample of 200-300 um thickness has caused changes in the time position and peak-to-peak amplitude of the first spike and the appearance of irregular second spikes in signals of the slow-speed sensor. Co-registration of acoustic signals in the skin sample with two sensors, the reference hydrophone and the malaria sensor, presented independently obtained evidence of the signals associated only with parasites in skin. Unlike the hydrophone, the malaria sensor does not resolve signal spikes which are close in time. Instead, it reveals some effects of the integration of close spikes. These effects influence time and amplitude parameters of the first, largest, spike in the signal, whose amplitude and time position correlate to parasites in the skin. The similarity of signal shapes and signal metrics observed in three different studies-non-invasive human studies, water model of nanobubbles, and the skin model with human parasites —suggests that the non-invasive skin signals detected in the field in malaria-positive subjects were caused by vapor nanobubbles generated around parasites in the skin.
Metrics N nb and T nb are independent of the amplitude derived metrics described above, and can be used in addition to and/or instead of the amplitude-derived metrics. In some embodiments, the malaria sensor can have reduced distortions of the tail baseline or a flat tail baseline to make it easier to detect the second and/or tail spikes.
Optical excitation and acoustic detection of parasites in a liquid sample of whole blood will now be described. The liquid sample included water, human blood (whole) and human blood with 50 Plasmodium Falciparum parasites per microliter (the lower limit of the microscopy detection of parasites in blood). Static and flowing samples were studied in an Eppendorf tube. The flow was achieved by the pipette-induced mixing of the tube content during the signal collection.
The same laser pulse as above (220 ps, 671 nm, 15 μJ) was delivered via the optical fiber (with 105 or 50 μm fiber core) as a 60-pulse train. The 50-um fiber was used with a reduced laser pulse energy of 3.6 uJ in order to maintain the same optical fluence as the one at the exit of the 105-um fiber. Acoustic detection was performed using a reference hydrophone of 1.0 mm diameter (with a bandwidth of 10-12 MHZ (hemozoin), and a sensitivity of 0.5 V/MPa at 5 MHZ (hemozoin)). The hydrophone tip was located at 2 mm (approximately) distance from the source.
The water and blood signals were detected as a reference (
As shown in
As shown in
Comparing the signals in
In the flowing sample, the amplitude HI-N metrics, coupled with tailored time-window (1.25us) which excluded the background signal, and the amplitude threshold of 4 mV, resulted in zero HI and N values for intact blood and 0.29 and 0.24, respectively, for blood with Plasmodium Falciparum parasites at the density 50 p/uL.
The volume exposed to the laser pulse may influence the background signal metrics. A smaller fiber has resulted in a reduction of 4 to 12-fold in the amplitude of the background signal (see, e.g.,
In these experiments, one parasite density has been used, 50 p/uL. For water-diluted blood samples (with a factor of 20:1), the threshold of detection of the same Plasmodium Falciparum parasites was 0.01 parasite per microliter of suspension. This is equal to 0.2 p/uL in the whole blood, and is still below the detection threshold of regular PCR, microscopy, and RDT, the three standard methods for detecting parasites in blood in the clinical and laboratory settings.
Compared to the skin model, the human whole blood seems to better support the generation of vapor nanobubbles without damping their expansion and collapse, similar to those in water. Thus, non-invasive detection of parasites in skin may benefit from the presence of blood around the subcutaneous parasites, which is the case for substantially all micro-vessels in dermis, the smallest of which are 5-10 um in diameter. The smallest nanobubbles detected in the human whole blood had a lifetime of around 500 ns, which is close to the detection threshold of 300-400 ns lifetime in the water model. Reducing the laser-probed (exposed to the pump laser pulse) volume may improve the signal-to-background ratio in detecting parasite-generated nanobubbles, but may require increasing the number of probed (scanned) locations and hence the diagnostic time. In addition, diluted blood samples returned more nanobubbles than the whole blood due to deeper laser penetration.
A chicken breast model was used to analyze parasites in melanin-free tissue. The sample was prepared from manufactured chicken breast meat (which remained visually wet). To model the malaria infection, blood with parasites was injected with a needle to the depth of 0.5-1.0 mm (as was verified later by measuring a cross-section of the sample as shown in
As shown in
The other difference to the tissue model describe above was that nanobubble signals in this model were not always observed in response to the first laser pulse and sometimes were detected in the same location after tens of laser pulses (
Despite a much higher tissue depth, 500-1000 um in the chicken breast model compared to 250 um in the human dark skin model, nanobubble signals were stably detected in melanin-free chicken breast tissue. Therefore, the presence of melanin and associated opto-acoustic background appears to be a limiting factor in the detection of parasite-generated vapor nanobubbles in human skin. As shown in
In the tissue model with chicken breast and the injected human blood, malaria parasites generated vapor nanobubbles similar to those generated in the gold-water model, with symmetrical expansion and collapse and without damping effects observed in the human skin sample. Nanobubble signals were observed at parasites at a tissue depth of up to 1.0 mm. The difference between the chicken breast and human skin models can be explained by the differences in the mechanical properties of two tissue samples and, additionally, by the higher volume of liquid (blood around parasites) in the chicken breast model compared to that in human skin model.
In summary, malaria parasites were detected through the acoustic response of vapor nanobubbles (defined earlier as hemozoin-generated vapor nanobubble). Hemozoin-generated vapor nanobubbles were generated and detected in whole blood, human dark skin and chicken breast tissue models, and in human subjects with Plasmodium falciparum and Plasmodium vivax parasite strains in the field studies. The result was achieved through comparative studies in six different experimental systems (in addition to two previously studied systems, individual infected human red blood cells and infected animals).
For the liquid sample, the method detects down to 0.01 Plasmodium Falciparum parasite (gametocyte) per microliter of solution and 0.1 Plasmodium Falciparum parasite (trophozoa) per microliter of solution. For the dark human skin, the method detected residual single Plasmodium Falciparum parasites (gametocytes). For the non-invasive model with a dark skin sample, the method might not detect all parasites present in the skin because it could detect only relatively large nanobubbles with the lifetime above 0.7 us. Such a high detection threshold suggests that smaller vapor nanobubbles are being generated in the skin but cannot be detected with the current excitation and detection setups.
The human field and two skin model studies revealed the influence of the mechanical properties of skin, such as the high elastic modulus and viscosity, and plastic deformation, on the generation and collapse, and hence the detectability of (relatively large, around 10 um max diameter) vapor nanobubbles. Smaller nanobubbles may be less influenced by the above mentioned properties of the skin and hence may improve the detection of parasites.
In human dark skin, the background signal of melanin may have a 10-fold higher amplitude than that of a parasite-generated nanobubble. The suppression of the melanin signal and its separation (decoupling) from the nanobubble signal improve the diagnostic performance of non-invasive skin-based device.
Another approach to improve the diagnostic performance is to use a minimally invasive skin probing device with the optical fiber penetrating 200 um of the upper skin may suppress the melanin background and improve the optical excitation of nanobubbles.
Terms of orientation used herein, such as “proximal,” “distal,” “radial,” “central,” “longitudinal,” and “end” are used in the context of the illustrated embodiment. However, the present disclosure should not be limited to the illustrated orientation. Indeed, other orientations are possible and are within the scope of this disclosure. Terms relating to circular shapes as used herein, such as diameter or radius, should be understood not to require perfect circular structures, but rather should be applied to any suitable structure with a cross-sectional region that can be measured from side-to-side. Terms relating to shapes generally, such as “circular” or “spherical” or “semi-circular” or “hemisphere” or any related or similar terms, are not required to conform strictly to the mathematical definitions of circles or spheres or other structures, but can encompass structures that are reasonably close approximations.
Conditional language, such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements, and/or steps. Thus, such conditional language is not generally intended to imply that features, elements, and/or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without user input or prompting, whether these features, elements, and/or steps are included or are to be performed in any particular embodiment.
The terms “approximately,” “about,” and “substantially” as used herein represent an amount close to the stated amount that still performs a desired function or achieves a desired result. For example, in some embodiments, as the context may permit, the terms “approximately”, “about”, and “substantially” may refer to an amount that is within less than or equal to 10% of the stated amount. The term “generally” as used herein represents a value, amount, or characteristic that predominantly includes or tends toward a particular value, amount, or characteristic. As an example, in certain embodiments, as the context may permit, the term “generally parallel” can refer to something that departs from exactly parallel by less than or equal to 15 degrees.
While a number of variations of the disclosure have been shown and described in detail, other modifications, which are within the scope of this disclosure, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the disclosure. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed.
Furthermore, certain features that are described in this disclosure in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations, one or more features from a claimed combination can, in some cases, be excised from the combination, and the combination may be claimed as a subcombination or variation of a subcombination.
Features, materials, characteristics, or groups described in conjunction with a particular aspect, embodiment, or example are to be understood to be applicable to any other aspect, embodiment or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings) may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The protection is not restricted to the details of any foregoing embodiments. The protection extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination so disclosed.
For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. Not necessarily all such advantages may be achieved in accordance with any particular embodiment. Thus, for example, those skilled in the art will recognize that the disclosure may be embodied or carried out in a manner that achieves one advantage or a group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.
Some embodiments have been described in connection with the accompanying drawings. The figures are not drawn to scale where appropriate, but such scale should not be limiting, since dimensions and proportions other than what are shown are contemplated and are within the scope of the disclosed invention. Distances, angles, etc. are merely illustrative and do not necessarily bear an exact relationship to actual dimensions and layout of the devices illustrated. Components can be added, removed, and/or rearranged. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with various embodiments can be used in all other embodiments set forth herein. Additionally, any methods described herein may be practiced using any device suitable for performing the recited steps.
Although this invention has been disclosed in the context of certain embodiments and examples, the scope of this disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the invention and obvious modifications and equivalents thereof. Any system, method, and device described in this application can include any combination of the preceding features described in this and other paragraphs, among other features and combinations described herein, including features and combinations described in subsequent paragraphs. While several variations of the invention have been shown and described in detail, other modifications, which are within the scope of this invention, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the invention. Various features and aspects of the disclosed embodiments can be combined with, or substituted for, one another in order to form varying modes of the disclosed invention. Thus, it is intended that the scope of the present invention herein disclosed should not be limited by the particular disclosed embodiments described above, but should be determined only by a fair reading of the claims that follow.
Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57. Specifically, this application claims the priority benefit of U.S. Nonprovisional application Ser. No. 16/213,923, filed Dec. 7, 2018, U.S. Provisional Application No. 62/595,971, filed Dec. 7, 2017, and U.S. Provisional Application No. 62/666,011, filed May 2, 2018, the entirety of each of which is hereby incorporated by reference and should be considered a part of this specification.
| Number | Date | Country | |
|---|---|---|---|
| 62666011 | May 2018 | US | |
| 62595971 | Dec 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16213923 | Dec 2018 | US |
| Child | 18472047 | US |
