Patent Application
20070298487
In the embodiment illustrated in
In order to perform the assay, a signal is generated by the transponder 110. That signal is directed upon an assay that is placed on the surface of the transponder 100, such that the assay is in communication with the sample 160. In the embodiment illustrated in
The assays 152, 154, 156, and 158 are illustrated as rectangles placed upon the surface 105 of the transponder 100. The assays are positioned such that the light from LEDs 112, 114, 116, and 118 is incident on the backside thereof. The light from LEDs 112, 114, 116, and 118, incident on respective assays 152, 154, 156, and 158, is reflected therefrom and back on to respective photodiodes 122, 124, 126, and 128. Consequently, in this embodiment, the light reflected from assay 152, 154, 156, and 158 and incident upon respective photodiodes 122, 124, 126, and 128 provides an indication of the assay response to the sample 160.
In order for the transponder 100 to detect a change in the assay, there must be a discernable difference in the light reflected from the assay 152, 154, 156, and 158 when the assay has indicated a positive response to the sample compared to a negative response to the sample. As previously defined, a positive assay response indicates a sample condition at or above a threshold level or amount. A negative assay response indicates a sample condition below a certain threshold level or amount. Examples of various assays are described in detail below.
In one embodiment, if the assay exhibits a positive response to the sample, the assay 152, 154, 156, and 158 (or the patch on which the assay is formed) changes color. For example, as bacteria grow in the sample 160 over time, the bacteria give off carbon dioxide. Certain dyes change color in response to increased concentrations of carbon dioxide. The change in color obviously changes the wavelengths of light that are reflected/absorbed by the assay. Therefore, if the light reflected from the assay changes, that change will be detected by photodiodes 122, 124, 126, and 128. Photodiodes 122, 124, 126, and 128 emit an electrical signal indicative of the assay's positive response. That change in wavelength is communicated to respective integrated circuits 132, 134, 136, and 138.
The transponder in the illustrated embodiment has provided therein capacity to perform four separate assays. These assays are completely distinct, with their own LED (e.g. 112), photodiode (e.g. 122), integrated circuit (e.g. 132), and assay (e.g. 152). These assays can be the same or different, and each assay response is detected and communicated independently of the others. However, other embodiments are contemplated wherein multiple assays are interrogated using the same LED's (e.g. 112) and photodiodes (e.g. 122). Also, it is contemplated that all transponder circuitry could be placed on one integrated circuit (e.g. 132) obviating the need for multiple chips to accommodate multiple assays.
The assays 152, 154, 156, and 158 can be bonded, imprinted, molded, screened, vapor deposited or otherwise attached to specific sites on transponder surface 105. In the illustrated embodiment, the assays 152, 154, 156, and 158 are optically interrogated by respective LEDs 112, 114, 116, and 118. Therefore, the housing 135 is a material that is optically transparent to the wavelengths of light emitted by LEDs 112, 114, 116, and 118 and detected by respective photodiodes 122, 124, 126, and 128. Specific optically transparent materials such as glass are described in detail below. It should be noted that the material need not be optically transparent at all wavelengths, but the material does need to be optically transparent at the target wavelength(s). The optical transparency enables the light from the LEDs 112, 114, 116, and 118 to propagate through the housing material 135 and be incident on respective assays 152, 154, 156, and 158. This also permits the light reflected from assays 152, 154, 156, and 158 to propagate through the housing to be detected by respective photodiodes 122, 124, 126, and 128.
In one example, the fourplex assay illustrated in
The specific material selected for the housing 135 for transponder 100 will depend upon the specific assay or assays being performed by the transponder and the target wavelengths for those assays. In the embodiments of the present invention where the assay is optically interrogated by sensors embedded within the transponder 100, the housing material 135 will be selected to be optically transparent at those wavelengths of light that will be used to detect a change in the assay 152, 154, 156, and 158 in response to the exposure of the assay to sample 160.
In the present invention, many different assays are contemplated as suitable. The types of assays that reside on the transponder can be virtually any biological, biochemical, immunological, nucleic acid, proteomic, genomic or other assay used in the medical diagnostic field. The assays themselves are conventional and not described in detail herein except as they are configured to operate with the transponder assay described herein. Biological and biosensor assays are described in U.S. Pat. No. 6,699,719 to Yamazuki et al. and U.S. Pat. No. 5,866,433 to Schalkhammer et al., which are incorporated herein by reference. Biological sensor arrays are described in Hanson, K. L., et al. “Biomolecules and Cells on Surfaces—Fundamental Concepts” in Microarray Technology and Its Applications, (U. R. Muller and D. V. Nicolau eds.) and Stekel, Dov, Microarray Bioinformatics, Section 1.2 “Making Microarrays” (Cambridge University Press 2005) which are incorporated by reference herein.
In addition to the optical test sensing technique employed in the embodiment illustrated in
Returning to the embodiment illustrated in
It is advantageous if the transponder 100 is not required to have a battery. In
The signals transmitted from photodiode 122, 124, 126, and 128 and converted into electrical signals for transmission to respective integrated circuits 132, 134, 136, and 138 are modulated onto an RF carrier wave and transmitted using antenna 145. In the embodiment illustrated in
Referring to
The received signal is conveyed to the radio frequency (RF) receiver 202. In certain embodiments, the receiver has an analog to digital (A/D) converter that converts the received RF signal to a digital signal. In other embodiments, the signal undergoes A/D conversion in the microcomputer. From there the signal is conveyed to the power supply 203. As previously noted, the power supply 203 is specifically one or more storage capacitors which can be used as a source of power for the components in the transponder. The power supply 203 is used as a source of power for the microcomputer 204 (in other embodiments the LEDs can have their own power source). Although only one microcomputer 204 is illustrated in
As previously noted, many different assays are contemplated. One example is a small, thin patch of sensor chemicals that is disposed on the outer surface of the transponder. This patch is sensitive to being either oxidized or reduced by the solution in which the transponder is placed. When testing biological samples, it is common to use a growth media in which to stimulate growth of certain microorganisms that may be present in the sample. The growth media is used to amplify the presence of the target organisms in order to facilitate detection.
In one embodiment of a chemical sensor patch, such a patch changes color depending upon the presence or absence of, for example, oxygen ions. When the concentration of oxygen ions in the sample exceeds a certain threshold amount, the chemical patch will change color. The color of the patch is interrogated by light generated from the light emitting diode mounted on the electronic circuit board within the transponder housing. The light emitting diode is configured such that it directs light onto the chemical patch. If the patch responds to an increase in oxygen concentration by changing color, then the light absorbance of the patch changes. This results in a change in the light that is reflected back to photodiode sensors on the electronic circuit board.
The photodiode measures the amount of light reflected from the patch. The photodiode generates a voltage that is proportional to the amount of detected light to a microcomputer mounted on the same circuit board. The voltage is measured by the microcomputer and a coded digital signal representing this value is transmitted by RF frequencies to a receiving device. As previously described, the receiving device is at some distance from the transponder and external to the environment in which the transponder is placed.
In yet another embodiment, the assay is a fluorescent indicator. Fluorescent indicators are well known to those skilled in the art and employed in a variety of biological assays. Briefly, the intensity of a fluorescent assay varies depending upon the environment in which the fluorescent indicator is placed. In one example, the intensity of the fluorescence of a chemical patch changes as a function of the acidity of the environment in which the assay patch is placed. Such a sensor could be used in conjunction with the LED and photodiodes described in previous embodiments. That is, light from the LED is directed onto the fluorescent indicator assay patch. In response to the light incident thereon, the patch emits fluorescent light. A fluorescent signal indicative of the acidity of the environment is emitted from the patch. This fluorescent signal is directed to the photodiode. The photodiode converts the fluorescent signal into an electrical signal. The electrical signal indicates the degree of intensity of the fluorescent signal, which in turn is indicative of the patch's response to the environment in which it is placed.
An example of an immunoassay patch is illustrated in
FRET is a technique for measuring interactions between two molecules (e.g. two proteins in vivo, antibody-antigen). In this technique, two different fluorescent molecules (first and second fluorophores) are genetically fused to the two molecules of interest. Regular (non-FRET) fluorescence occurs when a fluorescent molecule (fluorophore) absorbs electromagnetic energy of one wavelength (the excitation frequency) and re-emits that energy at a different wavelength (the emission frequency). Conceptually, each fluorophore has a two-peaked spectrum in which the first peak is the excitation peak, and the second is the emission peak.
For the combined FRET effect, the emission peak of the donor fluorophore must overlap with the excitation peak of the acceptor fluorophore. In FRET, light energy is added at the excitation frequency for the donor fluorophore, which transfers some of this energy to the acceptor fluorophore, which then re-emits the light at its own emission wavelength. The net result is that the donor emits less energy than it normally would (since some of the energy it would radiate as light gets transferred to the acceptor instead), while the acceptor emits more light energy at its excitation frequency (because it is getting extra energy input from the donor fluorophore).
Sources of background noise, or cross-talk, occur because (1) the donor radiates slightly (but not optimally) at the acceptor's emission wavelength, and (2) the acceptor is excited somewhat by the donor's excitation wavelength. Both of these will cause a non-FRET signal at the emission wavelength of the acceptor that needs to be modulated. There are two ways of doing this. One is to express each fluorophore individually in the same conditions in vivo in which FRET will be performed, and measuring this cross-talking. Thus, one would want to measure how much energy the donor radiates at the acceptor emission wavelength, as well as measuring how much the acceptor can be excited by the donor's excitation wavelength. A second, and easier, control to perform is to photobleach the acceptor fluorophore (by overwhelming it with light at its excitation frequency) to “knock out” its activity. This eliminates the energy transfer from donor to acceptor and typically causes an increase in the emission from the donor (due to the fact that it is not transferring energy to the acceptor). This increase of donor emission due to photobleaching of the acceptor is known as “dequenching” and allows one to determine how much the donor fluorophore is radiating at the acceptor emission frequency.
The benefit of FRET technology is that it has excellent resolution. The physics of the FRET energy transfer between donor and acceptor (which is non-radiative) is such that the efficiency falls off with the sixth power of the distance between molecules. Thus, FRET typically occurs when the two fluorophores are within 20 Å-100 Å (0.002 μm-0.01 μm) of each other, which means that the fluorophores must be brought together via very close molecule-molecule (e.g. protein-protein) interactions. Since molecules in biological samples can be about 50 Å to about 200 Å in diameter, the position of the fluorophores within the protein complex is critical. If the fluorophores are over 200 Å apart while the proteins to which they are fused interact with each other, no signal will be observed. Often, FRET experiments are done with just the putative interaction domains of the two proteins under examination because of the potential distance between the fluorophores in this environment. Complete molecules would likely cause the fluorophores to be too far apart for FRET to be observed.
Referring to
In this embodiment, the electrodes 650, 651 and 652 are electrically connected (via wires 662, 661 and 660 and contacts 622, 621 and 620, respectively) to an integrated circuit 625 mounted on the circuit board 610 inside the transponder 600. The change in current from patches 650 and 651 is detected by the integrated circuit 625. Electrode 652 is the reference electrode and, as such, its conductivity does not vary with the environment. Any change in the conductivity of electrodes 650 and 651 is measured against the reference electrode. The integrated circuit 625 generates a signal in response to the detected decrease in current. In turn, a signal indicating the measured result is modulated onto an RF carrier and transmitted by the transmitter (not shown). The transmitted signal is received by a device (not shown) remote from the transponder 600.
In yet another embodiment, the present invention is used in conjunction with continuous monitoring blood culturing instruments such as the Bactec™ instrumented blood culture systems from Becton, Dickinson and Company. In such systems a blood sample is introduced into the blood culture bottle which, typically, has growth media disposed therein. Many different media are employed depending upon the culture that is desired. Examples of media include blood culture media, aerobic/F media, etc. In the Bactec™ type of system, the blood or other sample is introduced into the blood culture bottle with the media therein. The blood culture bottle is then placed in a device for continuous monitoring of the sample. Such monitoring may take hours or days until a change in the sample is observed. The present invention is advantageous because such continuous monitoring is potentially not required. Rather, the transponder can be placed in the blood culture bottle with the media and the sample and, when a change in the sample is observed by the assay and transmitted to the circuitry within the transponder, that result can be transmitted to a remote station. That same signal can be used to indicate that the assay is complete. In the present invention, this embodiment is described with reference to
Referring to
One example of a pH sensitive dye is bromcresol purple. As bacteria grow in the sample they give off carbon dioxide, which changes the pH of the sample. The pH change causes the dye to change from its basic form (which absorbs light at a primary wavelength of about 590 nm) into its acid form (which does not strongly absorb at this wavelength). This results in a decrease of light absorption at about 590 nm. Placing an LED 415 near the chip 430 mounted on the interior surface of bottle 410, allows the chip to be optically interrogated by a light source external to the bottle. If the LED emits light at or near the 590 nm wavelength (a filter can be used to limit the wavelength of light transmitted onto the assay), a change in light absorption by the bromcresol purple, caused by the color change, can be detected. Specifically, the 590 nm wavelength light will be absorbed up to the point of which the pH change to the acid detectably lowers the dye's ability to absorb light at or near this wavelength. When the dye absorbs measurably less light at this wavelength, the light is able to transmit through the dye and strike the photosensor 445 of the chip underlying the dye. The resulting current causes the chip 430 to respond by transmitting an RF signal indicating the positive response of the assay to the change in the sample's pH. The transmitted RF signal is detected by a receiver placed near to the chip but outside the culture bottle 410.
A variety of different dyes can be used. These dyes can be selected to work with a particular LED or LED/light filter combination. The following table lists several dyes and their maximum light absorption in the basic form. These dyes are listed by way of example, and not by way of limitation.
The pK values are also listed for these dyes. The pK value is the pH at which 50% of the dye is in the base form and 50% of the dye is in the acid form. For a carbon dioxide sensor for bacterial growth, it is advantageous if the pK is between about 6 and about 8 (i.e. near a neutral pH of 7). Examples of dyes that meet this criterion include chlorophenol red, bromcresol purple, bromthymol blue and phenol red.
The dye can be affixed to the chip in a variety of ways. In one embodiment the chip is provided with an overcoat of glass to protect the electronic components from coming into contact with the sample that is being evaluated. The dyes are covalently attached to the glass using a silicon attachment chemistry. Specifically, the silicon will have an amino (NH2), hydroxyl (OH) or acid functional groups which can be used to attach the dye thereto. Such mechanisms for attaching dyes to substrates are well known in the pH paper field where hydroxyl groups are commonly used to attach dyes to paper.
Another method for attaching the dye to the chip is to use a sol-gel layer containing the dye. The sol-gel with the dye is placed on the glass surface, to which it adheres. The sol-gel can be composed of, for example, a mixture of tetraethoxysilane (TEOS) and phenoltrimethoxysilane (PTMOS). These two components, typically in a two-to-one molar ratio, also have a dye such as bromcresol purple dissolved therein. The two silanes form a sol-gel that allows water and carbon dioxide to enter. One method for preparing a sol-gel is described in Andreou, V., et al. “Determination of Pesticide in Water using Fiber-Optic Biosensors Based on Immobilized Enzymes,” Environ Sci. & Pollut. Res. (Special Issue), Vol. 3, pp. 290-291 (2002), which is hereby incorporated by reference.
In this embodiment the chip provides only binary information. Specifically the chip only indicates whether light does or does not transmit through the dye. Therefore, in this embodiment the chip does not provide enough information to generate a growth curve for the target substance in the sample. Specifically the chip cannot convey the amount by which the concentration of the target substance in the sample increases over time. However, a dye can be selected that reflects a different threshold concentration of the target substance (e.g. carbon dioxide) in the sample. That is, dye A might change color (thereby going from absorptive for a particular wavelength to transmissive for that wavelength) at a first concentration and dye B may change color at a second, higher, concentration. An increasing concentration of carbon dioxide in the sample can be tracked in this manner.
In an alternative embodiment, the surface of the chip is modified by attaching a bacterial signature DNA sequence thereto. Instead of the dye-based approach described in the previous embodiment, this embodiment uses a capture sequence of DNA on the transponder surface to test for the presence of target bacteria in the sample. In contrast to the previous embodiment, in this embodiment the chip has an optically transparent coating to which the capture sequence of DNA is coupled. The photodetector initially detects the optical signal incident thereon (i.e. before the assay exhibits a positive response to the sample, it is optically transparent). The assay transponder chip is placed in the sample bottle in contact with the sample therein. Bacteria to be identified is lysed and added to the sample. A detector sequence of DNA coupled to an optical probe dye is also added to the sample. As the bacteria in the sample attaches to the capture DNA on the transponder surface, detector sequences of DNA with the suitable optically opaque dye probe affix to the bacterial DNA. As an increased number of dye probes attach to the surface of the transponder, the surface becomes optically opaque to the incident light. At some point, due to the reduced amount of light transmitted through the transponder surface to the underlying photodetector, the electrical signal from the photodetector goes from on to off. This indicates that a certain threshold concentration of bacteria in the sample has been reached. Since, in this embodiment, the signal goes from on to off, the absence of signal indicates to the receiver that the threshold concentration of the bacteria in the sample has been obtained. Since power is required for transmission, and the transponder is powered by the light received from the photodetector, no transmission from the chip occurs in this off-state. The lack of signal indicates positive assay response in this embodiment. In these embodiments, it is preferable to configure the device such that, at least initially, the device is on. That way the transition from an on state to an off state can be properly interpreted as a positive response. If the device is configured to be in the off state initially, then it is difficult to determine whether the continued off state is attributable to the assay response or a faulty device.
It is also contemplated that the transponder of the present invention might be used for in vivo or in vitro testing. For in vivo testing, the transponder is implanted in a patient for detecting some physiological condition. For example, the assay on the surface of the transponder could be configured to detect elevated glucose levels in the patient. Such an assay could be used to monitor a diabetic patient. The assay would be reversible so that the transponders would not have to be constantly removed and replaced.
The transponder would periodically interrogate the assay to determine the patient's glucose level. The results of the analysis would be transmitted from the transponder to a remote receiver previously described. In this way, a patient could self-interrogate their glucose level using a wireless handheld device without the need for drawing blood for this analysis. This would provide a huge advantage to a diabetic patient who avoids monitoring their glucose levels because of the need for painful “sticks” in order to determine blood glucose levels.
The invention could also be used for in vitro testing in environments where a test sensor might adversely affect the local environment. For example, in large bioprocessing containers the transponder could monitor the viability of cell culture lines expressing Human Growth Hormone or recombinant bacteria expressing influenza vaccine. The sensor patch could employ a cell viability indicator such as diazoresor-cinol (e.g. Rezazurin). Rezazurin is a commercially available metabolic activity indicator. In its oxidized form the substance is light purple and in its reduced form is light pink. A transponder with such an assay on the surface thereof is configured to detect the change from its oxidized form (deep purple) to its reduced form (light pink) and communicate the fact of the color change for transmission to a remote device.
If cell viability became compromised, further testing of the bioprocessing container environment by other sensor patches or transponders (monitoring temperature or pH, for example) would prompt the supervisor of the bioprocessor to modify the environmental conditions accordingly.
In the embodiment described above where the assay is configured to selectively bind methicillin resistant staphylococcus aureus (MRSA), the assay changes color as bacterial cells have multiplied on the site. An antibody is used to bind these bacterial cells to the assay. The sensor, either internal to the transponder or external to a chip on which the assay is formed, spectroscopically measures changes in the color (e.g. light absorption) of the assay. Depending upon the complexity of the system used to make the measurement, the system may simply indicate that the surfaces change from one where light at the selective wavelength is transmitted through the surface to one where the light is mostly absorbed by the surface. In such a system, the output is essentially binary (that is, either yes or no). In other embodiments, the measurements are more complicated and the information obtained from these measurements is accordingly more detailed. For example, different values of transmissivity through the surface of the assay might be measured, which could indicate increasing concentrations of the target substance in the sample.
In yet other embodiments, the RF transponder with associated assay described above can be combined with a magnetized material such as ferrite powder and polymer resin. Such a biotransponder is moveable using an externally applied magnetic field. The transponder could be a part of a magnetic stirring element used to agitate the growth media in the sample. This would ensure that the sample is adequately mixed during measurement. Thus, a result obtained is more likely to represent the condition in the entire sample as opposed to an isolated portion of the sample that happened to have a higher or lower concentration of the target substance than in other portions of the sample.
One advantage of the RFID transponder assay described above is the fact that the chip in the transponder contains a unique address. Since the chip has a unique address, and that address can be read upon interrogation, there is no need to have a barcode on the label of the sample bottle.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.
