Abstract
A method of detecting a pathogen in a sample. The pathogen from the sample is captured with at least one recognition element. The sample is introduced to a paper-based microfluidic device having spaced electrodes disposed thereon. An impedance magnitude of the sample is measured across the spaced electrodes to detect a presence of the pathogen in the sample. A related paper-based microfluidic device and system are also disclosed.
Claims
1. A method of detecting a pathogen in a sample, the method comprising: capturing the pathogen from the sample with at least one recognition element on a substrate of a paper-based microfluidic device; washing the sample to remove electrically conductive solution; lysing the pathogen to release at least one electrically conductive chemical entity inside the pathogen; and measuring an impedance magnitude of the sample across a pair of spaced electrodes comprising silver and disposed on the paper-based microfluidic device to detect a presence of the pathogen in the sample.
2. The method of claim 1, wherein the sample is blood or plasma, the pathogen is a virus, and the at least one recognition element is an antibody.
3. The method of claim 1, wherein the at least one chemical entity comprises one or more of ions, proteins, antigens, enzymes, and biomolecules inside the captured pathogen.
4. The method of claim 1, wherein Triton x-100 is used to lyse the pathogen to release at least one electrically conductive chemical entity inside the pathogen.
5. The method of claim 1, wherein measuring an impedance magnitude of the sample to detect the presence of the pathogen in the sample includes measuring an impedance magnitude of a control sample, measuring the impedance magnitude of the sample and determining the change in impedance magnitude from the control sample to the sample to detect the presence of the pathogen in the sample.
6. The method of claim 1, wherein the impedance magnitude is measured at 1000 Hz and 1 V.
7. The method of claim 1, wherein detection of the pathogen includes the ability to selectively detect the pathogen in the presence of another type of pathogen.
8. The method of claim 1, wherein the paper-based microfluidic device having the pair of spaced electrodes disposed thereon has a microfluidic channel spanning the distance between the pair of spaced electrodes.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) FIGS. 1A and 1B illustrate paper-based microchips with flexible graphite and silver electrodes, respectively. FIG. 1C illustrates a paper-based microchip with a blood sample filling the channel between the pair of spaced electrodes.
(2) FIG. 2 shows a 3D schematic of the paper-based biosensing system.
(3) FIG. 3A illustrates impedance magnitude and FIG. 3B illustrates phase spectra of lysed HIV-1 subtypes (subtypes A, B, C, D, E, G, and panel) for frequencies between 100 and 1 MHz measured in the paper-based microfluidic device. Control samples were prepared by mixing streptavidin-coated magnetic beads conjugated with anti-gp120 antibodies (Biotin) with 1% Triton X-100 without any viruses in the sample. The viral loads of HIV-1 subtypes A, B, C, D, E, G, and the panel were 1.74×10.sup.8, 1.2×10.sup.8, 1.17×10.sup.8, 2.9×10.sup.8, 8.39×10.sup.8, 6.53×10.sup.8, and 1.49×10.sup.9 copies/mL, respectively.
(4) FIG. 4A illustrates impedance magnitude of HIV-1 lysate samples (subtypes A, B, C, D, E, G, and panel) at 1,000 Hz and 1 V. FIG. 4B illustrates normalized impedance magnitude change of HIV-1 lysate samples with respect to control samples. There was no virus in control samples, which was prepared by mixing the streptavidin-coated magnetic beads conjugated with anti-gp120 antibodies (Biotin) with 1% Triton X-100. “*” indicates statistically significant impedance magnitude change compared to all other groups. Statistical assessment on the results was performed using ANOVA with Tukey posthoc test for multiple comparisons. Statistical significance threshold was set at 0.05, p<0.05. Error bars represent standard deviation of the mean (n=3). The viral loads of these HIV-1 subtypes were 1.74×10.sup.8, 1.2×10.sup.8, 1.17×10.sup.8, 2.9×10.sup.8, 8.39×10.sup.8, 6.53×10.sup.8, and 1.49×10.sup.9 copies/mL for subtypes A, B, C, D, E, G, and panel, respectively.
(5) FIG. 5 illustrates the repeatability of the measured impedance magnitude for HIV-1 subtypes A, B, C, D, E, G, and panel. The minimum repeatability that calculated for these measurements was 88%.
(6) FIGS. 6A and 6B show a specificity evaluation of the paper-based microchip device. FIG. 6A illustrates average impedance magnitude and FIG. 6B illustrates phase spectra for lysed HIV-1 subtype A, Epstein-Barr virus (EBV), and mixture of HIV and EBV for frequencies between 100 Hz and 1 MHz. There was no virus in control samples. The average impedance magnitude spectra of lysed HIV and mixture of HIV and EBV was lower than the impedance magnitude spectra of control and EBV samples. There was no significantly difference between average impedance magnitude of control and EBV samples. Control samples were prepared by mixing streptavidin-coated magnetic beads conjugated with anti-gp120 antibodies (Biotin) with 1% Triton X-100 without any viruses in the sample. The viral loads of HIV-1 subtype A and EBV were 1.74×10.sup.8, 1.9×10.sup.9, copies/mL, respectively. The viral load of the mixture of HIV and EBV sample was 1.6×10.sup.8 copies/mL.
(7) FIG. 7A illustrates the impedance magnitude of the lysed HIV-1 subtype A, EBV, and mixture of HIV and EBV samples at 1,000 Hz and 1 V. Error bars represent standard deviation of the mean (n=3). Brackets connecting individual groups indicate statistically significant impedance magnitude. Statistical significance threshold was set at 0.05, p<0.05. FIG. 7B illustrates repeatability of the measured impedance magnitude for control, HIV-1 subtype A, EBV, and mixture of HIV and EBV samples. The minimum repeatability that calculated for these measurements was 79%.
(8) FIG. 8 shows normalized impedance magnitude change of HIV-1 lysate samples with respect to control samples at 1,000 Hz and 1 V in spiked blood and plasma samples. There was no virus in control samples, which was prepared by mixing the streptavidin-coated magnetic beads conjugated with anti-gp120 antibodies (Biotin) with 1% Triton X-100. Statistical assessment on the results was performed using ANOVA with Tukey posthoc test for multiple comparisons. Statistical significance threshold was set at 0.05, p<0.05. Error bars represent standard deviation of the mean (n=3). The viral loads of the HIV-1 subtype C used here was 1.17×10.sup.8.
(9) FIG. 9 shows fluorescent images of GFP tagged HIV-1 captured on the surface of the streptavidin-coated magnetic beads conjugated with biotinylated anti-gp120 antibody taken using a 10× objective lens.
(10) FIG. 10 shows Scanning Electron Microscopy (SEM) images of silver electrodes on paper-based microfluidic devices. SEM images were taken at 5 KV accelerating voltage and 7.6 mm working distance in FIG. 10A, and 7.3 mm working distance in FIG. 10B. Magnification was 103× in FIG. 10 A and 282× in FIG. 10B and signal was InLens.
(11) FIG. 11 shows the effect of time on the impedance magnitude of Triton x-100. Impedance magnitude of 1% Triton x-100 samples were measured at 1,000 Hz and 1 V every 20 minutes for 80 minutes. Statistical assessment on the results was performed using ANOVA with Tukey posthoc test for multiple comparisons. Statistical significance threshold was set at 0.05, p<0.05. There was no significant difference between the impedance magnitudes of the samples over the 80 minutes period (n=3, p>0.05).
(12) FIG. 12 shows average impedance magnitude spectra of control samples and lysed HIV-1 subtype C (1.17×10.sup.8) spiked in blood in FIG. 12A and plasma in FIG. 12B for frequencies between 100 and 1 MHz. Control samples were prepared by mixing streptavidin-coated magnetic beads conjugated with anti-gp120 antibodies (Biotin) with 1% Triton X-100 without any viruses in the sample. Average impedance magnitude of control and HIV-1 subtype C lysate spiked in blood is shown in FIG. 12C and plasma in FIG. 12D at 1,000 Hz and 1 V. Error bars represent standard deviation of the mean (n=3). Brackets connecting individual groups indicate statistically significant impedance magnitude difference. Statistical significance threshold was set at 0.05, p<0.05.
(13) FIG. 13 illustrates the average impedance magnitude of the HIV-1 lysate (subtype D) with viral load of 2.9×10.sup.6 copies/mL at 1,000 Hz and 1 V. The sample volume was increased to 5 mL for this virus concentration to generate a detectable impedance magnitude change compared to control samples. Error bars represent standard deviation of the mean (n=3). Brackets connecting individual groups indicate statistically significant impedance magnitude. Statistical significance threshold was set at 0.05, p<0.05.
(14) FIG. 14 illustrates equivalent electronic circuit and microchips with (a) a 2-probe electrode configuration and (b) a 4-probe electrode configuration.
(15) FIG. 15 illustrates impedance measurement results using the 2-probe and 4-probe electrode configurations. FIG. 15A shows impedance magnitude of the PBS/Triton samples with dilution factors between 0.05% to 1%. The impedance magnitude of samples with different dilution factors of PBS and Triton x-100 were measured at 1,000 Hz and 1 V. FIG. 15B shows the impedance magnitude shift measured by 2-probe and 4-probe configurations in the samples with different electrical conductivities. The impedance magnitude change measured in the samples was significantly higher when 4-probe configuration was used compared to 2-probe configuration.
DETAILED DESCRIPTION OF THE INVENTION
(16) Turning now to FIG. 2, the basic process flow for using the paper-based biosensing system to detect HIV-1 subtypes is schematically illustrated according to one exemplary method. In step (a) of the process, the HIV-1 viruses are captured using streptavidin-coated magnetic beads conjugated with gp-120 antibodies in whole blood sample. In step (b), the sample is washed to remove electrically conductive solution. In step (c), the captured viruses are lysed using 1% Triton x-100 to release ions, proteins, antigens, enzymes, and any biomolecules inside the captured viruses. These biomolecules change the electrical conductivity of the solution. In step (d), the viral lysate samples (now fully separated from the magnetic beads) are introduced to the paper-based microfluidic device with silver/graphite electrodes for impedance measurement of the viral lysate samples. An enlarged view of the paper-based microfluidic device is illustrated in FIGS. 1A and 1B, which also illustrates that the paper-based microfluidic device and its electrodes are flexible. The presentation of a blood sample is illustrated in FIG. 1C, in which blood has entered an opening on the top surface of the microfluidic device and has been transported through the channel between the electrodes such that the blood sample runs from one of the electrodes to the other of the electrodes. It is noted that in FIG. 1C, a pair of spaced electrodes are illustrated in a specific configuration. However, it is contemplated that other quantities or arrangements of electrodes or other electrode configurations might be used to measure impedance of a sample. For example, an array of finger electrodes, which are also called interdigitated electrodes, can be used for sensing.
(17) Specific examples are now provided of testing samples using this outlined process and the results thereof. These examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following example and fall within the scope of the appended claims.
Example I
(18) For the following example, the following reagents were obtained and used. Triton X-100 (100%), glycerol (100%), and Bovin Serum Albumin (BSA, 10%) were purchased from Sigma-Aldrich® (St Louis, Mo.). Dulbecco's Phosphate Buffered Saline (DPBS, 1×) was bought from Life Technologies' (Grand Island, N.Y.). HyPure™ Molecular Biology Grade Water was obtained from Fisher Scientific (Agawam, Mass.). Ethanol (200 proof) was bought from Sigma Aldrich® (Sheboygan, Wis.). Biotinylated polyclonal goat anti-gp120 antibody (4 g/mL) was obtained from Abcam® (Cambridge, Mass.).
(19) Magnetic beads conjugated with anti-gp120 antibodies were prepared. Streptavidin-coated magnetic beads (1 μm diameter) were purchased from Thermo Scientific (Rockford, Ill.). After diluting in DPBS by 1:10 (v/v), the beads were washed three times with DPBS (1.5 mL) using a BioMag® multistep magnetic separator from Polyscience Inc. (Warrington, Pa.). Antibody conjugation was done by adding biotinylated anti-gp120 antibodies with the stock concentration of 15 μg/mL to the magnetic beads solution and incubated for 2 hours at 4° C. on a rotator with a speed of 30 rpm for 2 hours. The non-conjugated antibodies in the solution were then removed from the sample through washing with DPBS (three times).
(20) HIV-1 was then captured on the magnetic beads conjugated with the anti-gp120 antibodies. Multiple HIV-1 subtypes [A, B, C, D, E, G, and panel (A, B, C, D, and circulating recombinant forms, CRF01_AE and CRF02_AG)] were mixed with conjugated magnetic beads and incubated for half an hour at room temperature on a rotator (15 rpm). The viral load of the HIV-1 subtypes in culture media were established to be 1.74×10.sup.8 (subtype A), 1.2×10.sup.8 (subtype B), 1.17×10.sup.8 (subtype C), 2.9×10.sup.8 (subtype D), 8.39×10.sup.8 (subtype E), 6.53×10.sup.8 (subtype G), and 1.48×10.sup.9 (panel) copies/mL. To prepare the control samples, DPBS solution without viruses was mixed with conjugated magnetic beads.
(21) The mixture of HIV-1 and conjugated magnetic beads were washed four times with 20% glycerol (diluted in grade water) to remove un-captured viruses in the solution and electrically conductive media. After the washing step, 1% Triton-X 100 (diluted in grade water) was mixed with the conjugated magnetic beads and incubated for 5 minutes to lyse the captured viruses. The viral lysis step compromises the membrane of the virus and releases the membrane phospholipids and proteins, capsid proteins, intracellular ions, retroviral enzymes, and nucleic acids into the background solution.
Example II
(22) Microchips were fabricated in the following manner. Two rail electrodes were patterned on hydrophobic transparency papers using silver ink from Engineered Materials Systems, Inc. (Delaware, Ohio). The electrodes geometry and design were cut on a double-sided-adhesive film (DSA) using a VLS2.3 laser cutter from Universal Laser Systems Inc. (Scottsdale, Ariz.). This DSA used as a mask and taped on top of a paper and conductive ink (graphite or silver) was poured on top of the mask to fill the openings on the DSA. A glass cover slip was used to distribute the ink evenly everywhere in the openings. The paper with the inks were then baked in an oven at 80° C. for one hour. After the ink dried, the protective DSA was removed and the electrodes patterned in the openings of the mask were left on the paper. The thickness of the electrodes was approximately 50 μm. The width and spacing of these two rail electrodes were 2 mm and 1 mm, respectively.
(23) FIG. 10 shows Scanning Electron Microscopy (SEM) images of silver electrodes on the paper-based microfluidic devices. SEM images were taken at 5 KV accelerating voltage and 7.6 mm working distance in FIG. 10A, and 7.3 mm working distance in FIG. 10B. Magnification was 103× in FIG. 10 A and 282× in FIG. 10B and signal was InLens.
Example III
(24) For the results that follow, Tukey's post-hoc test [Analysis of Variance (ANOVA)] was used to analyze the experimental results for multiple comparisons and the statistical significance threshold was set at 0.05 (n=3, p<0.05), unless otherwise indicated. Statistical analyses were performed with Minitab software (V14, Minitab Inc., State College, Pa., USA).
Example IV
(25) The viral lysate samples were introduced into the paper-based microfluidic device with two rail electrodes.
(26) To detect the captured HIV-1, the impedance magnitude and phase spectra were evaluated for viral lysate samples of multiple HIV subtypes including A, B, C, D, E, G, and panel over a range of frequencies between 100 Hz and 1 MHz at 1 V.
(27) These results are illustrated in FIGS. 3A and 3B. The impedance magnitude spectra of the viral lysate samples for all HIV subtypes were below the impedance magnitude spectra of the control samples for frequencies between 100 Hz and 100 kHz. The impedance magnitude of the viral lysate samples for frequencies above 100 kHz was same as the impedance magnitude of the control samples. Control samples were prepared by mixing DPBS and conjugated magnetic beads without HIV. Impedance phase spectroscopy of the viral lysate samples was same as control samples. The maximum impedance magnitude shift for all HIV subtypes with respect to control samples was observed to occur at 1,000 Hz. Therefore, this frequency was chosen to further statistically compare the impedance magnitude results.
(28) FIG. 4A shows the impedance magnitude of the viral lysate samples as well as control sample at 1,000 Hz and 1 V. The impedance magnitudes of the viral lysate samples were significantly lower than the impedance magnitude of the control sample (n=3, p<0.05). No significant difference was observed between the impedance magnitudes of multiple HIV subtypes (n=3, p>0.05). The impedance magnitude of the viral lysate samples were also normalized with respect to the impedance magnitude of control sample at 1,000 Hz as shown in FIG. 4B. The impedance magnitude shifts for HIV-1 subtypes A, B, C, D, E, G, and panel were 23±3, 32±3, 22±5, 28±3, 34±6, 28±1, 35±4, respectively.
(29) These results show the ability of the paper-based microchip to detect multiple HIV-1 subtypes spiked in DPBS at viral loads around 10.sup.8 copies/mL.
Example V
(30) The repeatability of these electrical sensing measurements were evaluated following the repeatability definition:
(31)
in which “I.M.” is the impedance magnitude of the viral lysate sample or control sample. As illustrated in FIG. 5, the repeatability of the impedance magnitude measurement for control sample and viral lysate samples were between 88% and 99%.
Example VI
(32) The specificity of the paper-based microchip for HIV-1 detection was also evaluated in the presence of Epstein-Barr virus (EBV) as a virus model. The impedance magnitude (FIG. 6A) and phase spectra (FIG. 6B) of the HIV, EBV, and the mixture of HIV and EBV samples were measured and compared with control samples where there was no virus in the sample. As FIGS. 6A and 6B illustrate, the impedance phase spectra of all the samples were the same; however, the impedance magnitude of the HIV and the mixture of HIV and EBV samples were lower compared to control samples. The impedance magnitude spectra of EBV samples was not significantly lower than control samples.
(33) Based on the previous tests, 1,000 Hz was chosen as a single frequency for impedance magnitude comparison between the samples as maximum impedance magnitude shift was observed at this frequency.
(34) FIG. 7A illustrates that the impedance magnitude of HIV and the mixture of HIV and EBV samples were significantly different compared to control samples at 1,000 Hz. The impedance magnitude of HIV and the mixture of HIV and EBV was not significantly different (n=3, p>0.05).
(35) The repeatability of these impedance magnitude measurements were also evaluated and measured. As illustrated in FIG. 7B, the repeatability of the experimental measurements was between 79% and 99%.
(36) These results evidence the ability of the microchip to selectively detect HIV-1 in the presence of another type of virus.
Example VII
(37) FIG. 8 shows normalized impedance magnitude change of HIV-1 lysate samples with respect to control samples at 1,000 Hz and 1 V in spiked blood and plasma samples. There was no virus in control samples, which was prepared by mixing the streptavidin-coated magnetic beads conjugated with anti-gp120 antibodies (Biotin) with 1% Triton X-100.
(38) Statistical assessment on the results was performed using ANOVA with Tukey posthoc test for multiple comparisons. Statistical significance threshold was set at 0.05, p<0.05. Error bars represent standard deviation of the mean (n=3). The viral loads of the HIV-1 subtype C used here was 1.17×10.sup.8.
(39) These results indicate that the method described above could be used to detect HIV-1 in either blood or plasma.
Example VIII
(40) To establish that HIV-1 was in fact captured on the surface of the streptavidin-coated magnetic beads conjugated with biotinylated anti-gp120 antibodies, HIV-1 was tagged with a green fluorescent protein (GFP) and captured on the beads.
(41) FIG. 9 shows fluorescent images of green fluorescent protein (GFP) tagged HIV-1 captured on the surface of the streptavidin-coated magnetic beads conjugated with biotinylated anti-gp120 antibody taken using a 10× objective lens. Because the images in the application are only in black and white, arrows have been used to indicate the areas that appeared green in the image.
Example IX
(42) The impedance magnitude of Triton x-100 Was measured over time to ensure that the impedance magnitude of Triton x-100 did not vary with time (as such a relationship, if significant, could raise issues relating to the confidence with which the viral samples were detected). FIG. 11 shows the effect of time on the impedance magnitude of Triton x-100. Impedance magnitude of 1% Triton x-100 samples were measured at 1,000 Hz and 1 V every 20 minutes for 80 minutes.
(43) Statistical assessment on the results was performed using ANOVA with Tukey posthoc test for multiple comparisons. Statistical significance threshold was set at 0.05, p<0.05.
(44) There was no significant difference between the impedance magnitudes of the samples over the 80 minutes period (n=3, p>0.05).
Example X
(45) Further trials were run to establish whether the change in impedance magnitude observed above in blood was similarly observable in plasma.
(46) FIG. 12A shows the average impedance magnitude spectra of control samples and lysed HIV-1 subtype C (viral load of 1.17×10.sup.8) spiked in blood for frequencies between 100 and 1 MHz and FIG. 12 B shows average impedance magnitude spectra of control samples and lysed HIV-1 subtype C (1.17×10.sup.8) spiked in blood for frequencies between 100 and 1 MHz. Control samples were prepared by mixing streptavidin-coated magnetic beads conjugated with anti-gp120 antibodies (Biotin) with 1% Triton X-100 without any viruses in the sample.
(47) The average impedance magnitude of control and HIV-1 subtype C lysate spiked in blood is shown in FIG. 12C and plasma in FIG. 12D at 1,000 Hz and 1 V. Error bars represent standard deviation of the mean (n=3). Brackets connecting individual groups indicate statistically significant impedance magnitude difference. The statistical significance threshold was set at 0.05, p<0.05.
(48) These results indicate that there was a statistically significant difference between the impedance magnitude of the control blood samples and the blood samples spiked with HIV-1 and a statistically significant difference between the impedance magnitude of the plasma control samples and the plasma samples spiked with HIV-1
Example XI
(49) FIG. 13 illustrates the average impedance magnitude of the HIV-1 lysate (subtype D) with viral load of 2.9×10.sup.6 copies/mL at 1,000 Hz and 1 V. The sample volume was increased to 5 mL for this virus concentration to generate a detectable impedance magnitude change compared to control samples. Error bars represent standard deviation of the mean (n=3). Brackets connecting individual groups indicate statistically significant impedance magnitude. Statistical significance threshold was set at 0.05, p<0.05.
Example XII
(50) Electrode geometry and configuration may have a significant impact on the electrical sensing method to detect and quantify HIV viral lysate. In FIG. 14, two microchip designs are illustrated which are subsequently used to demonstrate the effect of microchip designs with different electrode configurations (2-probe vs. 4-probe) on the impedance spectroscopy of samples with various electrical conductivities. FIG. 14(a)(ii) shows 2-probe electrode configuration and FIG. 14(a)(i) shows the equivalent electronic circuit used for testing. FIG. 15(a)(ii) shows 4-probe electrode configuration and FIG. 15(a)(i) shows the equivalent electronic circuit used for testing.
(51) Impedance measurement using the 2-probe configuration takes into account the contact resistance (R.sub.C) between the electrode and the sample in microchannel, whereas the 4-probe configuration eliminates the effect of contact resistance in impedance measurement on-chip. Contact resistance can play a major role in impedance analysis specifically when the electrical conductivity of the sample is low. In the HIV lysate impedance measurements the conductivity is as low as 20 to 200 μS/cm, therefore the contact resistance in our measurements would be high. Impedance of the 2-probe configuration is equal to R.sub.s+R.sub.C1+R.sub.C2, whereas the impedance magnitude of the system in 4-probe configuration is equal to R.sub.S.
(52) With reference to FIG. 15, the experimental results demonstrated that the 4-probe configuration showed a higher impedance magnitude change in PBS samples with different dilution factors when it is mixed with Triton x-100. The impedance magnitude of 1% Triton x-100 was 3.59 and 3.87 MΩ, when it was measured with 2-probe and 4-probe configurations, respectively. The impedance magnitude of Triton x-100 mixed with PBS at 1% v/v dilution factor was 1.41 and 0.89 MΩ, when it was measured with 2-probe and 4-probe configurations, respectively.
(53) The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.
