Organic Toroidal Array Apparatus of Making for Direct And Reagent-free Sensing of the Endotoxin Activities of a Single E. Coli Cell

Abstract

The invented organic memristive and memcapacitive device comprises of arrayed cross-bar donut-shape toroidal matrix self-assembling membrane on an electrode mimicking mitochondria's double membrane and the direct electron-relay functions enables a bio-communication signal flow directly between endotoxin and the sensor at a single E. coli cell concentration under nature enzyme-free, antibody-free and reagent-free conditions. The sensor offers multiple functions for monitoring neuronal synapse pulse energy output under influence of Lipopolysaccharide (LPS) and other important biomarkers. The robust analytical performances of the device for monitoring endotoxins are important to human health.

Description

FIELD OF THE INVENTION

The present invention relates to the field of electrochemical sensors, in particular, to a device having both characteristics in memristor/memcapacitor for direct sensing of endotoxin activities of single E. Coli cell and other proteins in biological substances.

BACKGROUND OF THE INVENTION

Lipopolysaccharide (LPS) is a common endotoxin from E. Coli bacteria, and is the major source causing infectious diseases over 20 million people worldwide. LPS is a major contaminant found in commercially available proteins, and it is also the major contaminate in biological ingredients in drugs and injectables, because even small amount of endotoxin can cause side effects such as endotoxic shock, injury, and even death; therefore a strengthened standard of drug purity is needed. However, removing LPS from pharmaceutical products, for intravenous application to 5 endotoxin units (EU) per kg of body weight per hr, is a challenge to researchers who thought this standard is unachievable [1-2]. E. Coli bacteria covers 75% its outer layer membrane with gram-negative exdotoxin LPS, and it stimulates the host's immune response of cytokines [3-4]. Recently, researchers reported LPS penetrates the gut-immune-barrier (GIB) causing liver infection [5]; LPS leaking from the tight junction in the gut membrane into the blood stream cause many diseases, autism, obesity, diabetes, Alzheimer's, chronic pain, and inflammation [6-10]. Furthermore, LPS can break the blood-milk barrier into the milk and may cause harm, as reported from collected cow milk, which was compromised by LPS, and may have caused mastitis [11]. A recently published paper reported human milk offers an advantage to correlate positively with gut microbiota and to maintain healthy oligosaccharide (HMO) isomers which are specific to human milk and that are necessary in the newborn infant's gut in the first week [12].

A paramount challenge was put on the researchers and industry as a whole for improving the LPS detection methods with more simplified procedure, more accuracy and precision, faster, and more affordable options. Because previously, a lack of sensitivity associated with the protein interference plus time consuming antibody and tracer assays hampered the ability to realize the unmet goals and fulfill these needs.

It is a well-accepted fact that breast-feeding offers more benefits for human babies' growth in nutritional and immune defense over cow milk [13-15], and it has been strongly recommended, as published by the World Health Organization [15]. We found very few tests or sensors, if any, to assess the energy outcomes at different neuronal synapse frequencies, such as slow-wave-sleeping and fast gamma frequency, between breast-feeding using human milk as compared with feeding organic cow milk in the presence of LPS challenge. We believe that this testing is important because not only it will increase our knowledge, but also it will provide first hand convincing evidence for preferring human milk for feeding infants in regards to the energy requirement for mental and physical development of infants. Our goal for this project is to develop a nanostructured memcapacitor/memristor sensor for antibody-free, reagent-free direct measurement of pg LPS, and to assess the energy outcome comparing human milk with cow milk. The intention is that the memcapacitor/memristor device represents, in concept, a baby's single neuron to “feel” the energy gain or loss in the presence of LPS. This project is based on our prior experience in using the memristor/memcapacitor to mimic hippocampus-neocortex neuronal network circuitry [16-20].

Acetyl co-enzyme A (AcCoA) is a leading substrate in a large variety of enzyme-catalyzed reactions, such as for choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) [21-25]. Szutowicz's group emphasized that AcCoA is the key factor for the survival or death of cholinergic neurons in course of neurodegenerative diseases [25]. Ivan Gout's group emphasized that the level of AcCoA is crucial to early embryonic development [26]. AcCoA is a thioester derived from catabolism of all major carbon fuels. AcCoA may play a role in the energy production, metabolism, memory, cell proliferation and early childhood development, and it is central to biological acetylation reactions. AcCoA deficiency leads to many diseases, such as diabetes, cancer, coronary disease, autism, Alzheimer's, and sudden infant death syndrome. Abnormality of CHAT activity may lead to these diseases because CHAT represents the most specific cholinergic marker in the CNS [27-28] and the spatial temporal manifestation of CHAT has been examined at both the protein and mRNA levels in different tissues of various species [28].

Furthermore, reports revealed that the virus replications of West Nile virus (WNV), the neurotropic flavivirus that is transmitted by mosquito bites causing meningitis and encephalitis in humans [29], involved the carboxylation of AcCoA to malonyl CoA through AcCoA carboxylase [29]. Therefore, sensitive quantitation of the CHAT activity, in terms of monitoring the changes of substrate AcCoA in biological specimens, is on demand for monitoring and diagnosing various diseases.

Challenges exist for providing a non-enzymatic label-free, reagent-less detection device for the direct detection of AcCoA with rapid detection time, free specimen preparation, and pM high sensitivity are paramount in order to avoid time-consuming assays and protein interferences. Many native enzymatic methods reported to detect AcCoA have the concentration range between mM to aM, such as the CoA cycling method [23], the carbon radioactive tracer labeling method [30-31], and the gas chromatography-mass spectrometry method [32]. The HPLC antibody method can reach to 0.1 μM level of AcCoA [26]. In view of the drawbacks of these methods, none of these methods can provide adequate sensitivity in pM level and the short testing time needed for testing AcCoA inside of the mitochondria cell when newborns consume human milk compared with that of cow milk in order to monitor the quality of the milk for babies.

It is well accepted that breast-feeding offers more benefits for human babies' growth in nutritional content and immune defense support over that of cow milk consumption [13-15] and it is a strong recommendation published by the World Health Organization [15]. However, to actively pursue real-time monitoring of breastfeeding and obtain the preliminary data using an innovative device is not practically feasible now. The goal of this project is to develop a nanostructured memcapacitor/memristor sensor for antibody-free, reagent-less direct measure pM AcCoA at different frequencies to assess the energy outcome comparing human milk with cow milk without protein interference and in a real-time and sensitive manner. The memcapacitor/memristor device will represent, in concept, a human infant single brain neuron's ability to “feel” or sense the energy gain or loss that is due to the presence of AcCoA signaling with the biomimetic CHAT of the sensor membrane in a biological specimen. This project is based on our prior experience in memristor/memcapacitor to mimic hippocampus-neocortex neuronal network circuitry [16-20].

SUMMARY OF INVENTION

It is an object of the present invention to evaluate the immunological advantage of human milk vs. organic cow milk regarding the pHFO formation at LPS challenges.

The intention is that the memcapacitor/memristor device is a sensor that represents, in concept, a baby's single neuron which is able to “feel” and react to the energy gain or loss in the presence of LPS. Our focus will be to determine how the pHFO occurs with dosage changes of LPS and the frequency change from SWS to 200 Hz.

It is an object of the present invention to demonstrate the memristor/memcapacitor's immunological capability in a contour mapping, that is based on a dual quantitative measurement of LPS in amperometric/voltage method while showing the advantage of human milk over cow milk.

It is an object of the present invention to provide a new generation of organic memristor/memcapacitor with Biomimetic FGFR-1 function and in Biomimetic of CHAT function in direct electron-relay systems.

It is an object of the present invention to provide a new generation of organic memristor/memcapacitor that is capable for dual sensing of functioning of AcCoA and LPS in single cell using milk specimen in current and voltage change without using antibody, mediator, labels and tracers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an art model for the 3D toroidal memcapacitor.

FIG. 2A depicts the 3D AFM image of the nanostructured islands membrane on a 50 nm gold chip of TCD/PEG/PVP and β-CD copolymer without o-NPA. FIG. 2B depicts the 2D AFM of the nanostructured islands membrane on a 50 nm gold chip of above membrane.

FIG. 3A depicts the art model of the SAM molecular polymer architecture for mimicking FGFR-1 in the presence of o-NPA. FIG. 3B depicts the art model of the SAM molecular polymer architecture for mimicking active sits of CHAT.

FIG. 4A illustrates the frequency affect on the hysteresis of the i-V curves of the FGFR-1 sensor in pH 7.0 PBS over scan rate from 1 Hz to 1 KHz without LPS. FIG. 4B depicts the CV profiles organic milk controls.

FIG. 4C depicts the CV profiles of human milk controls, and FIG. 4D depicts the CV profiles in 500 ng/mL LPS in human milk. In FIG. 4D, at 20 Hz, “a” curve depicts the milk sample spiked with a final concentration 50 ng/mL LPS in red; “b” curve depicts the control human milk sample; “c” curve depicts 500 ng/mL LPS spiked in the human milk sample in black.

FIG. 4E depicts the CV profiles in 500 ng/mL LPS in organic milk on the hysteresis of the i-V curves over 1 to 300 Hz. At 20 Hz, “a” curve depicts the milk sample spiked with a final concentration 500.0 ng/mL LPS in black; “b” curve depicts 50.0 ng/mL LPS spiked in the organic milk sample in red.

FIG. 5A depicts the CA curve profiles in organic milk samples with spiked LPS covered concentration from “a” to “h” with LPS at 0.0, 5.0 pg/mL, 50.0 pg/mL, 5.0 ng/mL, 50.0 ng/mL, 125.0 ng/mL, 250.0 ng/mL and 500 ng/mL. Each sample run triplicates.

FIG. 5B illustrates the CA profiles in organic milk samples with spiked LPS covered concentration from “a” to “e” with LPS at 0.0, 5.0 pg/mL, 50.0 pg/mL, 500.0 pg/mL, 50.0 ng/mL clearly showing at lower concentration range of LPS, the peak intensity is distinguishable between samples with LPS and the control. Each sample run triplicates.

FIG. 6 depicts the calibration plot curve of current density vs. LPS concentrations in organic milks samples using the CA method.

FIG. 7A depicts two different media solutions affecting on the single neuronal pulses profiles at 0.25 Hz; FIG. 7B depicts the voltage profiles in 40 Hz and FIG. 7C depicts the profiles in 250 Hz between pH 7.0 PBS buffer (red curve) and the human milk without LPS (black curve), respectively at ±10 nA, each sample run triplicates. FIG. 7D depicts the DSCPO profiles with or without LPS over LPS concentration ranges at 0.0 (a), 50 (b), 100 (c), 500 (d) and 1000 ng/mL(e) at 0.25 Hz at ±10 nA with each sample run triplicates. Insert is the curves for LPS at 500 and 1000 ng/mL, respectively. FIG. 7E depicts the DSCPO profiles with or without LPS over LPS concentration ranges at 50 ng/mL (a), 500 ng/mL (b), 1,000 ng/mL (c) and 0.0 ng/mL as the control (d) at 200 Hz, respectively, each sample run triplicates.

FIG. 8A depicts the quantitative calibration plot of LPS in human milk at 0.25 Hz and FIG. 8B depicts the plot at 200 Hz compared with LPS in organic milk at 0.25 Hz (FIG. 8C) and 200 Hz in FIG. 8D.

FIG. 9A compares the LPS effects on organic cow milk over 0, 50, 500 to 1000 ng/mL at 0.25 Hz and FIG. 9B depicts the voltage curves of organic mil at 200 Hz, respectively.

FIG. 10A and FIG. 10B depict the energy density (as Z) contour map and the image, respectively, are shown using human milk. The LPS concentration (as X) and synapse frequency (as Y).

FIG. 10C and FIG. 10D depict the energy density (as Z) contour map and the image, respectively, are shown using organic milk in the presence of the LPS concentration (as X) and synapse frequency (as Y).

FIG. 11A depicts the 3D energy distribution map vs. frequency and LPS concentrations in human milk. FIG. 11B depicts the 3D energy distribution map vs. frequency and LPS concentrations in organic milk.

FIG. 12 depicts the CV profiles in human milk with and w/o AcCoA.

FIG. 13 illustrates the CV curves in organic milk with and w/o AcCoA.

FIG. 14 illustrates the CA curve profiles in PBS pH 7.0 in the presence of 2 mM o-NPA.

FIG. 15 shows the calibration plot of current vs. AcCoA concentrations.

FIG. 16 depicts the extended plot of current vs. AcCoA concentrations from 2.0 pM up to 0.3 μM.

FIG. 17A depicts the voltage profiles of detections of spiked AcCoA in human milk compared with control at 0.25 Hz. FIG. 17B depicts the voltage profiles at 250 Hz.

FIG. 18A depicts voltage profiles of detections of spiked AcCoA in organic milk compared with control at 0.25 Hz. FIG. 18B depicts the voltage profiles of detections of spiked AcCoA in organic milk at 250 Hz.

DETAILED DESCRIPTION OF THE INVENTION

Example 1—Fabrication of the Nanostructure Self-Assembling Membrane (SAM) Gold Memristive/Memcapacitive Chips

The nanostructured biomimetic SAM was freshly prepared according to the published procedures based on cross linked conductive polymers of triacetyl-ß-cyclodextrin (TCD), polyethylene glycol diglycidyl ether (PEG), poly(4-vinylpyridine) (PVP) and ß-CD copolymer with appropriate amount of propositions on gold chip [21-22]. The chemicals were purchased from Sigma and went through purification procedures before use. A mixture of o-nitrophenyl acetate (o-NPA) in a molar ratio 1000:1 to the TCD mixture was incubated for 2 hrs at 35° C.; then the mixture was injected onto the gold surface and incubated for 48 hrs at 35° C. After that, we followed the clean procedures for completion of the SAM fabrication [21-22].

Example 2—Characterization of the Membrane

The morphology of the AU/SAM was characterized using an Atomic Force icroscope (AFM) (model Multimode 8 ScanAsyst, Bruker, Pa.). Data Collected in PeakForce Tapping Mode. Probes used were ScanAsyst-air probes (Bruker, Pa.). The silicon tips on silicon nitride cantilevers have 2-5 nm radius. The nominal spring constant 0.4 N/m was used. FIG. 1 illustrates an art model for the 3D toroidal memcapacitor. FIG. 2A depicts the 3D AFM image of horizontal conformational structure of the memristor/memcapacitor before the o-NPA was embedded on gold. FIG. 2B shows the 2D AFM image.

Example 3—Advantage of AcCoA's Rate Limiting Binding

Using the nano island structure SAM to mimic the function of Fibroblast Growth Factors Receptor-1 (FGFR-1) for improving fuel cell function was reported as shown in FIG. 3A [E. Chen's U.S. Pat. No. 8,632,925, Jan. 21, 2014]. It plays important roles in embryonic development, angiogenesis, wound healing, and malignant transformation (11). We thought using the function groups in the SAM membrane to mimic the AcCoA's human choline acetyletransferase (CHAT) binding sites intrinsically to mitochondria's double membrane compartment with the structure needed may be a simplified approach as a neuronal sensor model. The model of the device is to mimic CHAT's function in emphasizing of AcCoA's rate limiting step binding [1-5]. The possible electron-relay was proposed by the pyridine group in PVP, the COO⁻ group of TCD, the OH group from β-CD copolymer, and the carbonyl group from o-NPA through hydrogen bindings to be able to mimic s540, y552, c563, c550 and h324 of AcCoA binding sits in CHAT as Shown in FIG. 3B. The innovative approach is to first direct detect AcCoA in the mimicking binding sites of CHAT, without choline participates in the direct detection of AcCoA.

Example 4—Biomimetic Fibroblast Growth Factor Receptor 1 (FGFR1) SAM Membrane

FGFR1 is one of family receptors of tyrosine kinases. It plays important roles in embryonic development, angiogenesis, wound healing, and malignant transformation, bone development, and metabolism [35-36]. Y. Zhang's group reported mice with deleted FGFR1 exhibited an increased mobilization of endothelial progenitor cells (EPCs) into peripheral blood undergoing endotoximia, and the endotoximia was induced by injection of LPS [36]. Our project's initial step is to build a model device such that the device's SAM membrane mimics the FGFR1 receptor in the presence of LPS, which acts as a model metabolic product to access the FGFR1 function. By using this model to compare the effects of fresh human milk and organic cow milk at different frequencies of neuronal action/resting pulses at SWS and fast gamma frequency with or without LPS conditions to find out whether or not milk samples are energy efficiency or deficiency on the biomimetic brain cells will provide useful information to reveal which type of milk samples is immunologically advantage to infants. FIG. 3A shows the electron-relay system, and FIGS. 2A and 2B are the AFM images in 3D and 2D on a gold chip with the TCD/PEG/PVP/copolymer before adding o-NPA for embedding.

Example 5—Frequency Affects on Memristor/Memcapacitor's Performance

Evaluations of frequency's affect on memristor performance were conducted by Cyclic Voltammetric method (CV) in pH 7.0 saline solution at room temperature from a scan rate of 1 Hz to 1 KHz without using any biological specimen. Data are to be used for comparison between fresh human milk and USDA certified organic milk for infants with or without the presence of LPS covering the same range of real-time synapse action/resting potential pulses at different frequencies against controls.

FIG. 4A's i-V hysteresis curves were demonstrated with a switch point at the origin (0, 0) at almost all frequencies, except at kHz high frequency in the control medium PBS. When these perfect hysteresis behavior peaks occurred, especially at SWS frequency with a sensitive Direct Electron-Transfer (DET), and the switch point originates at origin, it indicates a healthy “newborn single neuron” exists before “feeding” it milk samples. Nonlinear frequency influence on current intensity is a characteristic of the memristor as reported in literature [9-12, 37-40]. FIG. 4B shows the controls in organic milk samples over 1 Hz to 1 kHz. The significant difference observed between the organic milk control, the PBS control, and the human milk control at SWS is that the organic milk did not have a butterfly type DET_red peak near −0.1 to −0.2 V, where as PBS and human milk had this peak and cross-points near the origin. Rather, the organic milk control had a strange DET_red peak at −0.595 V, and the control missed the cross-point near zero V. Further investigation is needed to determine what substances caused the unknown DET peak to occur. FIG. 4C is the human milk controls with well-defined sensory DET butterfly peaks crossed near the origin at SWS. FIG. 4D depicts that in human milk, 500 ng/mL LPS reduced the signal intensity at the SWS significantly. The LPS eliminated the original sensitive DET peaks, and that means the LPS first makes the neuron lose its sense of danger in the presence of toxins; this phenomenon matched our prior observations in the work of β-amyloidal (Aβ) that caused Alzheimer's sensory loss at SWS [9-12, 30, LPS, PSI]. In the worst case, the cow milk with LPS impaired heavily the DET sensory ability of our model neuron as compared to that in human milk, as shown in FIG. 4E.

Example 6—Quantitation of LPS Using the CA Method

Quantitation of LPS was conducted with two methods. The first was a Chronoamperometric (CA) method under two steps of fixed potential: −50 mV and −400 mV with each step duration of 100 ms, and the data rate is 20 kHz at room temperature under the conditions of antibody-free, radioactive tracer-free and reagent-free in certified organic milk for infants with seven LPS challenge levels from 5.0 pg/mL to 500 ng/mL against controls, each sample run triplicates.

The CA Method. The CA curve profiles were plotted using the biomimetic sensor in the presence of seven LPS concentration levels from 0, 5.0 pg/mL, 50.0 pg/mL, 5.0 ng/mL, 50 ng/mL, 125 ng/mL, and 250 ng/mL to 500 ng/mL against the control in organic milk samples as shown in FIG. 5A. FIG. 5B depicts the lower level LPS's response curves, showing more clearly the significant increase in signals at 5 pg/mL LPS over the control. The CA method for LPS produced a calibration curve with the regression equation y=−0.09+0.02×, n=21, S_y/x=0.23, r=0.998 with p<0.0001 covering the linear range from 5 pg/mL to 500 ng/mL in organic milk samples as shown in FIG. 6. The DOL result is 0.1 pg/mL per one cm² sensor in organic milk, i.e., by the CA method we are able to detect 5.0×10⁻⁴ EU E. Coli in 1 mL sample in 1 cm² sensor. Using this 0.031 cm² sensor, we are able to detect E. Coli cells in the range of 0.2-5 cell assuming 5 EU contains 2000-50000 E. Coli cells' activity [13, LPS, PSI]. The percentage of Pooled Relative Standard Deviation (PRSD %) of the organic milk samples over the entire linear range is 2.0%.

Example 7—Quantitation of LPS Using the Voltage Method

The second quantitation method was the voltage method, i.e., the DSCPO method, and the conditions were the same as described in the section of Assessing Energy Outcomes under Challenges of LPS by using human milk and organic cow milk samples under 4-5 LPS challenges from 50 ng/mL to 1000 ng/mL, respectively at ±10 A against controls at 0.25 and 200 Hz, respectively. Freshly obtained samples were without pretreatment. Human milk cooled by dry ice was delivered to the laboratory, and it was brought to room temperature naturally without any heating before spiking the LPS. All water used was autoclaved and double distillated from Fisher Scientific. LPS was purchased from Sigma, and it was dissolved in autoclaved and filtered PBS pH 7.0 buffers.

The Double Step Chronopotentiometry (DSCPO) method, as the voltage method, was used for assessing energy outcomes of slow-wave-sleeping (SWS) at 0.25 Hz and 200 Hz under the challenge of LPS at concentration ranges from 0, 50, 100, 500, to 1000 ng/mL of 4-5 levels with triplicates at ±10 nA, respectively. Samples were tested at each level without prior sample preparation, such as dilution or heating. The experiments were conducted at room temperature. The milk samples compared were human milk and USDA certified organic cow milk for infants, with and without LPS. Human milk was collected from a normal subject who breastfeeds a 1 month-old newborn (Lee Biosolutions Corp.). An electrochemical workstation was used (Epsilon, BASi, IN) with a software package from BASi. OriginPro 2016 (Origin Lab Corp., MA) was used for all statistical data analysis and figure plotting.

Assessing energy outcomes was conducted by comparing human milk and certified organic milk, both with and without LPS, at 0.25 Hz and 200 Hz, respectively, using the voltage method. FIG. 7A, 7B, 7C depict the synapse pulse control profiles using human milk, and compares samples using PBS media at 0.25, 40, 100, 200 and 250 Hz, respectively, without LPS. Curves overlap between the two media, and indicate human milk had no protein interference with the “single neuronal cell” as far as the neuron's energy output is concerned. FIG. 7D compares the signal intensity when testing human milk in the presence of various LPS concentrations at 0.25 Hz and the results show the signal intensity is inversely proportional to a wide range of LPS concentrations from 50 ng/mL to 1000 ng/mL at 0.25 Hz. At 50 to 100 ng/mL, the biphasic pulse shape integrity is maintained; however the insert curves show that at 500 and 1000 ng/mL, the biphasic pulses are destroyed, and the cell net voltage intensity is close to zero. FIG. 7E demonstrates a similar trend at 200 Hz, that the cell net voltage gets close to zero, at a higher concentration at 200 Hz, but at 50 ng/mL, the signal increases more than 30% compared with that at zero LPS. This is a bad effect, a negative outcome of wasted energy. FIG. 8A depicts the quantitative calibration plot of LPS in human milk at 0.25 Hz and FIG. 8B depicts the plot at 200 Hz compared with LPS in organic milk at 0.25 Hz (FIG. 8C) and 200 Hz in FIG. 8D. FIG. 9A compares the LPS effects on organic cow milk over 0, 50, 500 to 1000 ng/mL at 0.25 Hz and FIG. 9B depicts the voltage curves of organic mil at 200 Hz, respectively.

FIG. 8A illustrates the linear calibration curve at 0.25 Hz of volumetric energy density vs. LPS concentration range from 50 to 500 ng/mL using human milk, and it produced a linear regression equation Y=125-0.25×, r=0.9993 (n=12), P<0.0001, Sy/x=2.0. The Detection of Limits (DOL) is 0.3 ng/mL, i.e., in a 40 μL sample; it detects 12 pg LPS using a 1 cm³ sensor, our sensor is 3.11×10⁻⁷ cm³. Herein the DOL in our sensor is 3.73×10⁻¹⁸ g for LPS means we are able to detect a single E. Coli bacterium because an antigen is in 10⁻¹⁷ g range. At 0.25 Hz, the energy density ranges between 123.2 and 0.11 μWHr/cm³ using human milk specimens with an imprecision value 3.0% compared the energy range of 9.8 to −0.042 μWHr/cm³ for organic milk. FIG. 8B shows the nonlinear curve for LPS at 200 Hz using human milk. In contrast, FIGS. 8C and 8D show no sensitivity towards LPS over the same concentration range using organic milk. Human milk offers more than 12.5-fold high energy than organic milk and 100-fold sensitivity for LPS than organic milk.

Example 8—a Contour Mapping Method for Evaluation of Human Milk Immunological Advantage Under the LPS Challenge

The data obtained from the quantitation using the voltage method was used for evaluation of human milk immunological advantage under LPS challenges compared with that of the organic cow milk samples in 3D mapping method. The energy density results were put into the “y” column, the spiked LPS concentration over 0.0 to 1000 ng/mL was put into the “x” column, and the frequency was at the “z” column having two levels of 0.25 to 200 Hz. After converting the three data columns into a random XYZ correlation matrix, one can plot the contour maps and analyze the spatiotemporal formation of the pHFO, if it exists among human milk or organic milk samples. The real-time data obtained from the DSCPO method was converted to volumetric energy density, E=C_s·(ΔV)²/(2×3600), where C_s is the specific volumetric capacitance, C_s=[−i·Δt/ΔV]/L, C_s is in F/cm³ [33-34], Δt is the time in second, ΔV is the voltage in V, i is the current in Amps, and L is the volume in cm³.

The energy density contour maps associated with the images are presented in FIG. 10 with energy density as Z, LPS concentration as X, and discharge pulse frequency as Y. FIGS. 10 A and 10B depict the contour map of energy density vs. LPS concentration in the frequency change using the human milk samples and FIGS. 10C and 10D depict the contour map of energy density vs. LPS concentration in the frequency change using the organic milk samples. It is obvious that human milk produced tremendous higher energy (showing as the light in the image) with an intensity more than 10-fold higher, especially at SWS even in the presence of LPS.

Example 9—Evaluation of Immunological Advantage Under LPS Challenges

The comparisons of the immunological advantage under LPS challenges were evaluated through the study of the formation of the pHFO using a 3D energy density map method. The energy density results were put into the “y” column, the spiked LPS concentration over 0.0 to 1000 ng/mL put into the “x” column and the frequency was into “z” column having two levels of 0.25 to 200 Hz. After converting the three data columns into a random XYZ correlation matrix, one can plot the contour maps and analyze the spatiotemporal formation of the pHFO if a pHFO exists among human milk or organic milk samples. FIG. 11A depicts a 3D contour map of the relationship between energy density, LPS concentration and frequency using human milk. As we can see, at SWS, human milk held the highest neural synapse energy for with or without the presence of LPS challenge over the range from 0.0 ng/mL LPS to less than 200 ng/mL, until the LPS reaches 500 ng/mL, the energy gradually reduced to zero, in other words, human milk samples have an order of magnitude higher energy density at LPS=0 vs. organic milk at SWS and 200 Hz, respectively as shown in FIG. 11 A compared with FIG. 11B. At 0.25 Hz, 50 ng/mL LPS caused 100% energy reduce in organic milk vs. human milk only 6.25% reduced. The rate of LPS reducing synapse energy is 10-times faster in organic milk samples than in human milk samples. It is estimated from the map at LPS 5 EU/ng, human milk maintained 96.6% original energy strength vs. organic milk only 49% strength at 0.25 Hz.

Example 10—Potential Application in Superconducting

According to FIG. 4A, the device has potential application in superconducting device at zero-bias with 200 Hz scan rate in PBS solution with ±1 μA peak superconducting current. FIG. 5A, FIG. 5B and FIG. 14 demonstrated the sine curvatures with oscillation in both situations for with and without analyte, either LPS or AcCoA indicated the toroidal arrays configuration induced the amplified supercurrent at a finite applied V_dc potential (−600 mV) for LPS and (−200 mV) for AcCoA, respectively.

REFERENCES

• [1]. P. O. Magalhaces, A. Mlopes, P. G. Mazzolas et. Al., J. of Pharm Pharmaceutical Sci 10(3), 388-404, 2007.
• [2]. M. Daneshiam, A. Guenther, A. Wendel et. al, J. of Immunological Methods 313, 169-175, 2006.
• [3]. Y. A. Knirel, M. A. Valvano, Bacterial (Eds), lipopolysaccharides, structure, chemical synthesis and interaction with host cells, Springer, 2011.
• [4]. T. Reimer, M. Brcic, M. Schweizer, T. W. Jungi, J. Leukocyte Biology, 83, 1249-1257, 2008.
• [5]. G. I. Tsiaoussis, S. F. Assimakopoulos et. al., World J. of Hepatology, 7(17), 2058-2068, 2015.
• [6]. LPS: the switch for inflammation in the gut and beyond, www.realfoodforager.com
• [7]. A. I. Petra, S. Panagiotidou, E. Haziagelaki, et. al., Clinical Therapeutics, 37(5), 984-995, 2015.
• [8]. Anna Struncka (ed), Cellular and molecular biology of autism spectrum disorders, Prague University Press.
• [9]. I. M. Chiu, B. A. Heeesters et. al., Nature 501, 52-57, 2013.
• [10]. B. Petschow, J. Dore, P. Hibberd, et. al., Annals of the New York Academy of Sceinces, 1306, 1-17, 2013.
• [11]. M. Lehmann, O. Wellnitz, R. M. Bruckmaier, J. of Diary Science, 96(2), 889-896, 2013.
• [12]. M. Lorna, A. Deleoz, K. M. Kalanetra, et. Al., J. of Proteome Research, 14, 491-502, 2015.
• [13]. Chapter five of breast feeding, J. of Tropical Pediatrics, Mother and Child Nutrition in Tropics and subtropics, World Health Organization: Breastfeeding: A brochure for ready reference, WHO, Geneva, 1981.
• [14]. N. Khaldi, V. Vijayakumar et al., J. Of Food Chem. 62, 7225-7232, 2014.
• [15]. M. L. Marin, R. Avrovo et. al., J. Pediatric. Castroenterol. Nutrition 49(3), 343-348, 2009.
• [16]. E. T. Chen, J. Thorten, C. Ngatchou, S-H Duh, P. T. Kissinger, NSTi-NanoTech 2, 107-110, 2013.
• [17]. E. T. Chen, J. T. Thornton and C. Mulchi, Jr., Sensors and Transducers Journal, 191(8), 84-99, 2015.
• [18]. J. T. Thornton, C. Mulchi Jr, P. T. Kissinger, E. T. Chen, Sensors and Transducers Journal, 191(8), 100-113, 2015.
• [19]. E. T. Chen, J. Thornton, C. Ngatchou and S-H Duh, NSTi-Nanotech, 2, 169-172, 2014.
• [20]. S. H. Duh, J. T. Thornton, P. T. Kissinger and E. T. Chen, Biotech, Biomaterials and Biomedical: TechConnect Briefs, 172-175, 2015.
• [21]. U-M. Bilhardt, P. Stein and G. M. Whitesides, Bioorganic Chemistry, 17, 1-12, 1989.
• [22]. A. B. A. Prempeh, A. K. Prince and E. G. J. Hide, Biochem. J. 129, 991-994, 1972.
• [23]. T. Kato, J. of Neurochemistry, 42 (4), 903-909, 1984.
• [24]. A-R. Kim, R. j. Rylett, B. H. Shilton, Biochemistry, 45, 14621-14631, 2006.
• [25]. A. Szutowicz, H. Bielarczyk, A. Jankowska-Kulawy, T. Pawetczyk, A. Ronowsa, Neurochem Res, 38, 1523-1542, 2013.
• [26]. Y. Tsuchiya, U. Pham, W. Hu, S-I Ohnuma, I. Gout, PLOS One, 9(5), e97693, 2014.
• [27]. Y. Oda, Pathology International 49, 921-937, 1999.
• [28]. D. Wu and L. B. Hersh, J. Neurochemistry, 62, 1653-1663, 1994.
• [29]. T. Merno-Ramos, A. Vazquez-Calvo, J. Cacas, et. Al., Antimicrobial agent and chemotherapy, 60, 307-315, 2015. Doi:10.1128/AAC.01578-15.
• [30]. J. J. Kamphorst, M. K. Chung, J. Fan, J. D. Rabinowitz, Cancer and metabolism, 2(23), 1-8, 2014.
• [31]. W-P Lu, S. W. Ragsdale, J. Biol. Chem, 266(6), 3554-3564, 1991.
• [32]. M. Riera-Borrull, E. Rodriguez-Gallego, A. Hernandez-Aguilera et al., J. Am. Soc. Mass Spectrom, 2015. DOI: 10.1007/s1336-1-015-1262-3.
• [33]. E. T. Chen, J. Thornton, C. Ngatchou, S-H. Duh, P. T. Kissinger, NSTi-Nanotech, (3), 115-118, 2013.
• [34]. E. T. Chen and C. Ngatchou, Clean Technology (3), 204-207, 2011.
• [35]. M. Mohammadi, G. McMahon, L. Sun, C. Tang, P. Hirth, Science 276, 955-959, 1997.
• [36]. Y. Zhang, N. Su, F. Luo et. al., Int. J. Biol. Sci, 10(9), 1064-1071, 2014.
• [37]. J. Martinez-Rincon, M. D. Ventra, Y. V. Pershin, Physical Review B, 81(19), 195430-1-195430-7, 2010.
• [38]. M. D. Pickett, G. Medeiros-Ribeiro and R. S. Williams Nature Materials, DOI: 10.1038/NMAT3510, 2012.
• [39]. R. Kozma, R. E. Pino, G. E. Pazienza, Advances in neuomorphic memristor science and applications, Springer Publisher, 2012.
• [40]. M. D. Ventra, Y. V. Pershin, Nanotechnology 24, 255201, 2013.

Claims

1. A direct single endotoxin cell detecting device comprising: an electrode comprising a substrate of gold;a self-assembling membrane (SAM) comprising a polymer matrix comprised of an electrically conductive copolymer; wherein the copolymer is further comprised of:one or more first β-cyclodextrin molecules having at least one or more acetyl groups; one or more polyethylene glycol polymers; one or more poly(4-vinylpyridine) polymers; one or more β-cyclodextrin copolymers forming the SAM having a mitochondria-like surface structure comprising an array of nano islands; and the nano islands are vertically oriented and affixed on the substrate; an organic hydrophobic material forms an electron-relay network of (pnp)n or (npn)n with the function groups in the nano islands that mimics active sites of the electron-relaying of Fibroblast Growth Factor Receptor 1 (FGFR1) or choline acetyltransferase (CHAT).

2. According to claim 1, wherein the SAM of the device interacts with the organic hydrophobic material o-nitrophenyl acetate (o-NPA) formed an electron-relay network with TCD . . . PEG or TCD . . . PVP.

3. According to claim 2, wherein the device has a toroidal array dual sensing function of single molecule E. coli and Acetyl CoA.

4. According to claim 3, wherein the organic nanobiomimetic memristive/memcapacitive sensing apparatus have the nanometer air gap serving as the dielectric insulator between the electron-relay circuits.

5. According to claim 4, wherein the device directly measured and monitored endotoxin and its energy outcomes is Lipopolysaccharide (LPS) from E. coli in biological fluid under antibody-free, tracers-free, and reagent-free conditions using a double step chronopotentiometry (DSCPO) method, i.e., voltage method.

6. The use of the organic nanobiomimetic memristive/memcapacitive sensing apparatus according to claim 4, further includes procedures of applied a voltage or a current cross the MEA working electrode, as anode and a bare gold electrode as cathode, having another bare gold lead as the reference electrode, immersed in a biological media containing a target analyte, a changing currents flow or voltage change occurred in the sensor, that a signal intensity is recorded either in proportional or inversely proportional to the analyte concentration; wherein the target analyte can be detected and monitored by against the calibration curves.

7. According to claim 6, wherein the linear concentration range measured is up to 0.5 μg/mL in 40 μL specimen samples with a Detection of Limits (DOL) of 0.3 ng/mL having the energy density range between 123.2 and 0.11 μWHr/cm3 using human milk specimens at 0.25 Hz and ±10 nA with an imprecision value 3.0% (n=12) against 9.8 to −0.042 μWHr/cm3 for organic milk samples using the voltage method.

8. According to claim 6, wherein the device direct measures and monitors intrinsic pM level of AcCoA and its energy outcomes in the biomimetic mitochondria cell of a single neuron in real-time with wide-band synapse frequencies from SWS to fast gamma ripples for monitoring milk quality and deficiencies using the double step chronopotentiometry (DSCPO) method, i.e., voltage method.

9. According to claim 6, wherein the device direct measured LPS concentration with a Detection of Limits (DOL) result is 1.2×10−16 g LPS in 40 μL milk samples on a 0.031 cm2 sensor under antibody-free and reagent-free conditions using the Chronoamperometry (CA) method.

10. According to claim 6, wherein the device direct measured AcCoA between the range 2 pM to 0.3 μM by the CA method; it has a linear range from 2 pM to 0.4 nM. The value of Detection of Limits (DOL) is 1.2×10−12 M/cm2.

11. According to claim 9, wherein the device direct measured AcCoA with a recovery value of 103%, and the method produced an error of less than 2% (n=12) by using milk samples.

12. According to claim 6, wherein the device is able to distinguish healthy High Frequency Oscillation (HFO) and Pathological High Frequency Oscillation (pHFO) formations through a contour energy map linked to LPS concentration in biological fluid, energy density and frequency from 50 ng/mL up to 1000 ng/mL from Rapid Eye Movement (REM) sleep frequency, fast gamma frequency to Sharp Wave-Ripple Complexes (SPW-R) frequency.

13. According to claim 6, wherein the device is a memristive/memcapacitive device with the non linear hysteresis loops vs. scan frequencies observed and the spontaneous discharge characteristics were observed between 0.11 μWHr/cm3 to 123.2 μWHr/cm3 inversely related to the LPS concentrations.

14. According to claim 13, wherein the device has potential application in superconducting device at zero-bias with 200 Hz scan rate in PBS solution with ±1 μA peak superconducting current.