Antigen-Dissociation from Antibody-Modified Nanotransistor Sensor

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Antigen-Dissociation from Antibody-Modified Nanotransistor Sensor Arrays as a Direct Biomarker Detection Method in Unprocessed Biosamples Vadim Krivitsky,† Marina Zverzhinetsky,† and Fernando Patolsky*,†,‡ †

School of Chemistry, The Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel Aviv 69978, Israel Department of Materials Science and Engineering, The Iby and Aladar Fleischman Faculty of Engineering, Tel-Aviv University, Tel Aviv 69978, Israel

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‡

S Supporting Information *

ABSTRACT: The detection of biomolecules is critical for a wide spectrum of applications in life sciences and medical diagnosis. Nonetheless, biosamples are highly complex solutions, which contain an enormous variety of biomolecules, cells, and chemical species. Consequently, the intrinsic chemical complexity of biosamples results in a significant analytical background noise and poses an immense challenge to any analytical measurement, especially when applied without prior efficient separation and purification steps. Here, we demonstrate the application of antigen-dissociation regime, from antibody-modified Si-nanowire sensors, as a simple and effective direct sensing mechanism of biomarkers of interest in complex biosamples, such as serum and untreated blood, which does not require ex situ time-consuming biosample manipulation steps, such as centrifugation, filtering, preconcentration, and desalting, thus overcoming the detrimental Debye screening limitation of nanowire-based biosensors. We found that two key parameters control the capability to perform quantitative biomarkers analysis in biosamples: (i) the affinity strength (koff rate) of the antibody−antigen recognition pair, which dictates the time length of the high-affinity slow dissociation subregime, and (ii) the “flow rate” applied during the solution exchange dissociation step, which controls the time width of the low-affinity fastdissociation subregime. Undoubtedly, this is the simplest and most convenient approach for the SiNW FET-based detection of antigens in complex untreated biosamples. The lack of ex situ biosample manipulation time-consuming processes enhances the portability of the sensing platform and reduces to minimum the required volume of tested sample, as it allows the direct detection of untreated biosamples (5−10 μL blood or serum), while readily reducing the detection cycle duration to less than 5 min, factors of great importance in near-future point-of-care medical applications. We believe this is the first ever reported demonstration on the real-time, direct label-free sensing of biomarkers from untreated blood samples, using SiNW-based FET devices, while not compromising the ultrasensitive sensing capabilities inherent to these devices. KEYWORDS: Nanowire, field effect transistors, biomolecules, separation, preconcentration, biosensors, ionic screening, antibody

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their limited separation peak capacity and dynamic range of detection; therefore, the direct analysis of complex biosamples leads to seriously decreased sensitivities and assay reliabilities,11 along with dramatically increased complexity and detection times. Thus, the effective separation of biomolecules is critical to increase the dynamic range of detection, specificity, and sensitivity. Microfluidic systems have contributed significantly to the development of separation techniques for biosample analysis and exhibited efficient separation of DNA, particles, and proteins.12,13 The increasing popularity of microfluidic systems

he detection of biomolecules is critical for a wide spectrum of applications in life sciences and medical diagnosis. Biosamples are highly complex solutions, which contain a large variety of biomolecules and chemical species. For instance, blood represents a mixture of more than 10,000 different proteins, and an additional uncountable number of chemical species, with concentrations in a range varying over 10 orders of magnitude.1 Consequently, the intrinsic chemical heterogeneity of biosamples results in a significant background noise and poses an immense challenge to any analytical measurement, especially when applied without prior efficient separation and purification steps. Current protein analysis approaches, based on multidimensional sample separation2−10 that is on the use of various micro/nanoporous materials, coupled to mass spectrometry detection, fall short because of © 2016 American Chemical Society

Received: June 23, 2016 Revised: August 4, 2016 Published: August 31, 2016 6272

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Figure 1. Schematic illustration of the operation of an antibody-modified SiNW FET sensing device (upper part, green receptor units) versus nonimmune reactive protein-modified SiNW FET control device (lower part, red receptor units). (1) The SiNW FET sensing devices exposed to low ionic-strength sensing buffer, which calibrates both devices to baseline electrical signal. (2) With the introduction of a biosample, containing the target biomarker antigen, the conductivity of both devices increases in a similar manner, due to the high ionic content, which screens the target antigen association-related electrical signal. Thus, no analytical signal can be extracted from the association regime curve (upper and lower parts 2). (3) Fast wash-out of the sensing devices with the low ionic-strength sensing buffer reveals two distinct separable dissociation regimes. A slower dissociation rate of the target antigen from its specific antibody-modified SiNW FET sensing device can be observed (upper part 3), while a faster dissociation rate is observed for the control nonspecific device (lower part 3).

directly accomplish their potential sensing capabilities under physiological environments, relevant to many important biological, medical, and diagnostic applications, due to the high salt concentration of these samples, characterized by a Debye screening length of ∼1 nm, and the resulting screening of the charge-based electronic signals,40 and are therefore firmly restricted to detection under low salt concentration conditions ( koffnonspecific species, as expected. Importantly, this later dissociation zone, characterized by an electrical response higher than the sensing buffer baseline electrical response (Figure 5B, horizontal dashed blue line)

Figure 5. Concentration-dependent sensing of the CA 15−3 antigen in unprocessed serum samples using the dissociation regime mode approach, at a low flow rate of 1 chamber-volume exchange per minute. (A) The black, red, blue, and turquoise curves show the 6277

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enough for the clinical diagnostics of CA 15−3 in relevant physiological concentration range (greater than 67 pM).57 According to the previous experimental observations, and at a “flushing step” flow rate of 1 chamber volume exchange/min (see Figures 4 and 5), we can confidently assume based on the extracted koff rates that antibody species displaying dissociation rates in the range between koff = 5 × 10−3 and 1 × 10−8 s−1 will serve adequately as detection receptors. Importantly, this is the range for most commercially available monoclonal antibody species for biomedical applications. Antibody species exhibiting lower affinity constants will dissociate concurrently with the nonspecific low-affinity species (Figure 4) and fail to serve as appropriate receptors based on the dissociation-based detection method. Furthermore, through a controlled increase of the dissociation flow rate step, we can further narrow the nonspecific species dissociation subregime window so that antibodies of lower affinity may be used as well. It is important to emphasize that all dissociation-based experiments in this work were performed after achieving complete association of the antigen species, electrical signal plateau, to the surface of the nanowire sensing devices (as evidenced by prior experiments performed under low ionic strength conditions as discussed before). This is highly important if concentrationdependent analytical sensing results are required, since the final concentration of the antigen under test is calculated based on the difference between the baseline electrical signal under low ionic-strength conditions, prior to the antigen association, and the dissociation curve plateau achieved after complete removal of the low-affinity species from the vicinity of the sensing devices (high-affinity regime). Thus, changes in the amount of associated antigen species to the nanosensing devices, due to differences in the antigen-association time applied during the sensing experiments, will eventually cause changes to the extracted concentration-dependent calibration curve. For the purpose of future real world applications, a constant antigen association period of time, or alternatively a complete association allowed, must be applied for analytical consistency. Finally, for the purpose of real-world practical applications, there is a great need to quantitatively measure the exact concentration of a certain biomarker in the tested biosamples. It is important to emphasize that in our previous concentrationdependent measurements (Figure 5); in order to quantitatively assess the antigen concentration, we first measured the transition time between the dissociation subregimes by exposing the sensing devices to an antigen-free serum sample. This sample serves as reference for the extraction of the accurate time-point in which a complete removal of the lowaffinity species is achieved. After this point in time, an accurate quantitative assessment of the antigen concentration can be confidently performed, as demonstrated in Figure 5C,D, assuming slow dissociation of the antigen species occurs as shown before. Clearly, such required calibrating steps, along the requirement for an antigen-free reference biosample, make the detection scheme cumbersome, indirect, and probably inaccurate, mainly due to the inherent potential differences between the tested biosample and the reference antigen-free biosample. For this reason, we fabricated our sensing arrays consisting of two main types of chemically modified nanodevices (Figure 6), as follows: (i) the first group represents the sensing nanodevices, chemically modified with the antibodies specific against the antigens under examination, and (ii) the second group of nanodevices is chemically modified with a nonimmune reactive protein (or a nonspecific antibody

Figure 5. continued complete raw electrical response of a representative anti-CA 15−3modified SiNW FET sensing device to the association and dissociation (raising phase) of its specific antigen CA 15−3 in unprocessed bovine serum sample at concentrations of 0 (control antigen-free sample), 55, 135, and 535 pM, respectively. (B) The black, red, blue and turquoise curves show the normalized dissociation regime electrical response of the anti-CA 15−3-modified SiNW FET sensing device in unprocessed bovine serum sample at CA 15−3 concentrations of 0 (control antigen-free sample), 55, 135, and 535 pM, respectively. Each antigen sample was flowed for ca. 6 min (until reaching association plateau) before the washing-out with low-ionic strength sensing buffer was performed (sensing buffer-SB, 155 μM sodium phosphate buffer pH = ∼8.0). The black arrow indicates the time of solution exchange from the unprocessed serum samples to the low-ionic strength sensing buffer. (C) Concentration-dependent calibration curves of the CA 15−3 antigen extracted at different points of time along the measured dissociation regime curves in (B). The black squares, red circles, and blue, green, pink and brown triangles correspond to points in time depicted as black, red, blue green, pink and brown dashed lines in (B), respectively. The curves show that the analytically relevant high affinity regime, marked in turquoise circles, begins after 450 s along the dissociation curves in (B). The red circles depict nonanalytically relevant points in time along the dissociation regime curves. (D) Concentration-dependent calibration plot of the CA 15−3 antigen (at the analytically relevant time points marked by turquoise circles in (C)).

displays a strong and reproducible dependence on the concentration of the antigen protein CA 15−3 in serum, which remains tightly bound to the SiNW FET device surface. A further experimental evidence on the presence of the protein antigen confined to the surface of the nanodevices was demonstrated by the use of a regeneration buffer (glycine buffer, pH = 3), which rapidly brings the dissociation of the high-affinity antigen−antibody pairs and causes the electrical response of the nanodevices to return to their initial baseline electrical level, after the subsequent flow of the low ionicstrength sensing solution (see Supplementary Figure S4). This corresponds well with literature results demonstrating the use of “regeneration” buffers for the fast and complete dissociation of antibody−antigen pairs.54 Additionally, concentrationdependent experiments performed on untreated serum samples spiked with different concentrations of the CA 15−3 antigen reveal the robustness of this quantitative detection approach based on the simple examination of the dissociation regime window. Notably, as the concentration of CA 15−3 in the tested serum sample increases (Figure 5B), more of the biomarker molecules associate to the SiNW devices surface, leading to stable larger electrical response (in relation to the SB baseline response).50,55,56 The strong specific interaction between the anti-CA 15−3 and the antigen CA 15−3 allows the complete washing of the nonspecifically adsorbed salts and biomolecules within the sensing channel, performed by the fast flushing with low ionic strength sensing buffer, while maintaining the majority of surface-bounded CA 15−3 antigen molecules, in order to finally measure their sample concentration quantitatively. Therefore, monitoring the dissociation regime of antigens from antibody-immobilized surfaces, with high-affinity capabilities, allows performing direct analytical detection using SiNW FET-based device, without applying any sample manipulation steps. Importantly, this method allows performing quantitative protein measurements, sensitive 6278

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receptor) and serves as on-chip internal reference devices. This later group of devices, due to the absence of specific interactions with the antigens in the biosample under test, does only nonspecifically interact with low-affinity fast dissociating species present in the biosample, and allows the real-time simple extraction of the accurate transition time at which the first dissociation subregime is reached and a quantitative assessment of antigen concentration can be carried out. Thus, using these nonreactive on-chip reference devices allows for the simultaneous sensitive and quantitative detection of biomarkers in real time mode. In addition, as mentioned before, increasing the flow rate of the dissociation-related “flushing step” is expected to lead to a narrowing of the lowaffinity fast-dissociation subregime time window, and thus allow for a faster and more accurate quantitative detection of the antigen species. For this purpose we adopted the use of a microfluidic chamber of smaller dimensions (see Supporting Information for information on chamber dimensions), so as to allow for considerably higher nominal flow rates (chamber volume exchange rate), using flow rates easily achievable with our fluidic pumping system. Additionally, the use of smaller dimension microfluidic channels, instead of the previously used 100 μL larger chamber, is expected to lead to a more efficient fluid exchange during the dissociation washing regime, along with the critical requirement of considerably lower biosample volumes, possibly lower than a few microliters. Figure 6 demonstrates the measurements performed aimed at the detection of the CA 13−5 biomarker based on the differential on-chip detection approach discussed above, this time using a considerably higher effective flow rate of 100 μL/min (chamber volume exchange rate is 330 chamber volumes per minute, chamber volume 0.3 μL). By comparing the dissociation curves obtained from the nonimmune active protein-modified nanowire devices to the dissociation curves attained by the specificantibody-modified devices, it is readily possible to measure the amount of the biomarker associated with the antibody-modified nanowire-based devices, as demonstrated in Figure 6. The SiNW device modified with the protein BSA, which does not have specificity against the biomarker CA 15−3, reaches a plateau ∼25 s after the flushing of the sensing buffer (Figure 6, red and blue curves), with an apparent koff of ca. koff = 1.4 × 10−1 s−1 (see Supplementary Figure S5). However, the desorption kinetics from the antibody-modified SiNW device is considerably slower and correlates well with the concentration of the targeted antigen (Figures 5 and 6). Obviously, the application of a faster solution exchange rate dramatically narrows the fast-dissociation subregime window by a 10-fold factor, from ca. 250 s in previous measurements, to ca. 25 s under the faster flow conditions, thus allowing for a considerably faster detection cycle without the requirement of off-line calibration steps, while not affecting the quantitative and sensitive accurate assessment of the antigen biomarkers (see Supplementary Figure S4). These results clearly demonstrate that the simultaneous combination of bioreceptors of suitable koff values, along with the use of microfluidic chambers of adequate dimensions (that allow the fastest possible fluid exchange), allows for the direct fast, sensitive, and accurate detection of biomolecules based on their dissociation regimes. Also, this faster flow condition broadens the possibility to use antibody receptors, or other bioreceptors, of considerably lower affinity. Importantly, our proposed “dissociation-based” detection approach can be readily applied to untreated blood samples. Our measurements demonstrate that

Figure 6. Multiplex single-chip differential detection of the CA 15−3 antigen by the use of specific and nonspecific chemically modified SiNWs FET devices, at a high flow rate of 330 chamber-volumes per minute. (A) The blue curve shows the dissociation regime response of a 55 pM CA 15−3-spiked unprocessed serum sample from a control nonspecific BSA-modified SiNW FET device (not specific against the antigen CA 15−3). The red curve shows the dissociation regime response of a 250 pM CA 15−3-spiked unprocessed serum sample from a control nonspecific BSA-modified SiNW FET device (not specific against the antigen CA 15−3). The black curve corresponds to the dissociation regime response of a CA 15−3-spiked serum sample, at a concentration of 55 pM, from an anti-CA 15−3-modified SiNW FET sensing device. The vertical dashed red line indicates the beginning of the high-affinity regime, which refers to the point in time where all the low-affinity species are completely removed from the SiNWs FET environment, as clearly evidenced by the complete return of the nonspecifically modified on-chip devices to the original electrical response under low-ionic strength sensing buffer (sensing buffer, SB, 155 μM sodium phosphate buffer pH = ∼8.0). (B) Concentrationdependent calibration curve of the CA 15−3 antigen, extracted at the time point marked by the vertical red dashed line in (A) (beginning of the high-affinity regime). 6279

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Nano Letters a complete removal of the low-affinity nonspecific species (blood cells, proteins, salts, and small chemicals) is effectively achieved after a period of ca. 300 s, koffnonspecific = 1.6 × 10−2 s−1 (see Supplementary Figure S6, under low flow conditions, and lasting shorter, ca. 80 s under high flow conditions), which is considerably lower than the dissociation rate of the high-affinity antigen−antibody interactions. In conclusion, we have demonstrated the application of antigen-dissociation regime from antibody-modified nanowire sensors as a simple and effective direct sensing mechanism in complex biosamples, serum and untreated blood, which does not require ex situ time-consuming biosample manipulation steps, such as filtering, preconcentration, and desalting. We believe this is the first ever reported demonstration on the realtime, direct label-free sensing of biomarkers from untreated blood samples using SiNWs-based FET devices. We found that two key parameters control the capability to perform quantitative biomarker analysis from biosamples: (i) the affinity strength (koff rate) of the antibody−antigen recognition pair, which dictates the time length of the high-affinity slow dissociation subregime window, and (ii) the flow rate applied during the solution exchange dissociation step, which controls the time width of the low-affinity fast-dissociation subregime. Thus, the combination of high-affinity antibody receptors, along with high solution-exchange flow rates, leads to the effective deconvolution of the complex dissociation regime window into two fully separated dissociation subregimes, thus allowing the simple and accurate quantitative detection of protein biomarkers. Previous attempts to overcome the Debye length limitation utilizing SiNW FET-based technology required complicated procedures to reduce the dimensions of the antibody receptor,47 or the on-chip integration of components aimed at the desalting, preconcentration, and separation of the biomarker from the biosample.55 Undoubtedly, this is the simplest and most convenient approach for the SiNW FET-based detection of antigens in complex untreated biosamples, such as serum, urine, and blood. The lack of ex situ biosample manipulation time-consuming processes enhances the portability of the sensing platform and reduces to minimum the required volume of tested sample as it allows the direct detection of untreated biosamples (5−10 μL blood or serum), readily reducing the detection cycle duration to less than 5 min, while not compromising the ultrasensitive sensing capabilities inherent to SiNW-based FET devices, factors of great importance in near-future point-of-care medical applications.

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ACKNOWLEDGMENTS

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REFERENCES

This work was in part financially supported by the Legacy Fund, Israel Science Foundation (ISF).

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* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b02584.

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Materials and Methods section describing all experimental procedures in detail (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 6280

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