Development and Investigation of a Dual-Pad In-Channel Referencing

Aug 27, 2012 - ... University of Delaware, Newark, Delaware 19716, United States .... Biochimica et Biophysica Acta (BBA) - General Subjects 2018 1862...
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Development and Investigation of a Dual-Pad In-Channel Referencing Surface Plasmon Resonance Sensor Qiongjing Zou, Nicola Menegazzo, and Karl S. Booksh* Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States S Supporting Information *

ABSTRACT: Herein, we describe the construction of a novel dual-pad referencing surface plasmon resonance (SPR) sensor utilizing electrolytic grafting of diazonium salts to individually functionalize two gold pads positioned in a single fluidic channel. Using a dove prism, a simple single axis optical train may be employed without compromising the analytical performance. Once functionalized, one pad is used as the analytical sensing pad for detection of molecular interactions while the other serves as the reference pad, compensating for background refractive index fluctuations. The reference pad effectively compensates bulk refractive index changes and temperature variations as well as other nonspecific effects. The sensor was applied to calibration of a pH-responsive polymer layer in the presence of bulk refractive index and temperature variations. Monitoring selective attachment of a protein is also demonstrated. To our knowledge, this is the first implementation of inchannel referencing SPR sensor utilizing diazonium salt-based surface chemistry.

A

diazonium salt bond results in a reproducible and stable surface coating.1,3,7 In addition, the surface can first be modified by the diazonium salt followed by attachment of the protein, or the strategy can be turned around by first conjugating diazonium salt precursor to protein followed by diazotation to the perspective diazonium salt modified protein, enabling one-step attachment of bioreceptor-diazonium salt adducts,2,8,9 which can effectively eliminate nonspecific binding of protein to the surface. The drawback of the strategy is the reaction condition for diazotation may not be compatible with all proteins.10 In traditional SPR biosensing instrumentation such as the BIACore or SPReeta, a separate reference channel is employed to compensate for background effects like nonspecific binding and temperature fluctuations,11−13 though in practical fieldbased applications, compensation for thermal effects can be the major source of signal drift.14 This reference channel is oftentimes millimeter(s) away from the analytical channel and consequently resides in a different microenvironment. Employing a reference channel to account for temperature fluctuations is only as effective as the environment if the two channels are similar. The inability to controllably functionalize multiple unique SPR sensing pads within a single channel has limited the temperature stability of SPR sensors. Diazonium salt chemistry enables rapid and selective individual functionalization of multiple SPR sensing pads within a single channel, which provides a promising direction to better temperature compensation.

novel strategy for functionalizing the surface plasmon resonance (SPR) sensing pads that relies on electrolytic grafting of diazonium salts has been reported recently.1−3 The innovation of selectively electrografting different diazonium salt layers onto the gold surface enables independent functionalization of the reference and analytical SPR sensors within a single channel. SPR spectroscopy is a well-established surface sensitive optical technique. SPR sensors have been widely used in realtime and label-free detection of biomolecules with high sensitivity.4 SPR spectroscopy exploits the interaction of collective charge oscillations propagating parallel to a metal/ dielectric interface, so-called surface plasmon polaritons (SPPs), with the environment near the metal surface. The SPP’s field of effect decays exponentially from the metal surface with a typical penetration depth of less than 300 nm.5 Within this region, SPR is sensitive to any changes in the refractive index (RI), and as a result, SPR cannot differentiate between background effects (such as bulk RI changes or nonspecific binding) and specific binding events (i.e., antigen to antibody).6 This illustrates two major issues presently limiting SPR bioanalyses which need to be addressed: (1) fouling of the sensor surface and (2) temperature fluctuations. Typically, SPR biosensors rely on modification of surfaces with bioreceptors to selectively recognize (bio)molecules of interest. The influence from interfering species is mitigated by utilizing coatings that reduce levels of fouling of the sensor surface. The diazonium salt-based coating chemistry discussed in this contribution offers a particular set of advantages: diazonium salts can be electrografted in less than 1 min, the electrografting is real-time controllable with regards to electrochemical selection of surfaces to modify, and the covalent gold© 2012 American Chemical Society

Received: June 14, 2012 Accepted: August 27, 2012 Published: August 27, 2012 7891

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Scheme 1. Electrochemical SPR Instrument Using a Dove Prisma

a

(A) Analytical pad, (R) reference pad, (W) working electrode.

refractive index matching oil (RI = 1.5150), and collected with two 200 μm-diameter optic fibers. The light exiting the collection fibers entered an Andor Technology Shamrock 303 imaging spectrophotometer utilizing a TE-cooled and charge coupled device (CCD) camera. Depending on the RI range desired, the spectrophotometer covered the spectral range to 59.48 nm with a 1200 g/mm grating or 569.5 nm with a 150 g/ mm grating. A polyetheretherketone (PEEK) electrochemical flow-cell with an internal volume of ∼180 μL was constructed in-house. All three electrodes (counter, reference, and working electrodes) were connected to the potentiostat (Bioanalytical Systems). The Pt counter electrode and a chloridized silver quasi reference electrode were immersed in the chamber, though only one gold pad was connected with the working electrode. (See Scheme S-1 in Supporting Information.) Sensor Modification Procedure. Prior to the electrografting procedure, the diazonium salt (4-phenylacetic diazonium chloride, 4-PAD) used in this study was generated by the in situ method according to the procedure previous published.3 (See Supporting Information for detailed procedure.) The 4PAD solution formed was then immediately used to perform electrografting by syringe injecting the solution into the 180 μL electrochemical flow-cell. A series of five 2 s, −400 mV pulses were applied only to the analytical gold pad. A diazonium salt layer with an exposed, reactive carboxyl group was obtained (Scheme 2). The diazonium salt coated layer was then modified

An alternative to diazonium salts, microspotting of individual sensing pads with unique coatings, could be employed to create SPR arrays with reference sensors within a single channel. Corn and co-workers15−19 and Cheng and co-workers13,20,21 focus on SPR imaging, an array format technique, on planar waveguides. Corn is interested in developing thiol-based surface chemistries for high throughput screening of biomolecular interactions by SPR spectroscopy, whereas Cheng’s group is combining SPR with microfluidic sample handling for determining biomolecule binding affinities. However, the multiple wash, rinse, and dry steps make microspotting not readily or rapidly performed in a closed channel format; in many research or noncommercial applications, it is important that bioreceptor functionalization on a microfluidic sensor chip can be prepared freshly in realtime on an ad hoc basis. Herein, we describe the fabrication and investigation of the first dual-pad in-channel referencing SPR sensor to illustrate compensation for bulk RI changes and temperature fluctuations, as well as other nonspecific effects. Placed side-by-side, the two sensors encounter nearly identical temperature and field effects. The analytical SPR sensing pad measures interactions between the analyte and selective coating as well as undesired effects. The reference SPR pad with a nonactive surface (i.e., one to which the analyte will not interact) is exposed to the same solution and thereby only measures the undesired effects. Those effects can then be subtracted away to leave only data relevant to association and dissociation processes.

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Scheme 2. Diazonium Salt Generation and Electrografting

EXPERIMENTAL SECTION

Experimental Setup. The gold-coated dual-pad slides were prepared according to the procedure previous published.22 A minor difference is a Scotch tape strip was pressed onto the middle of the slide and peeled off after sputtering to leave a 1 mm wide clear line between the two gold-coated pads. (See Supporting Information for detailed procedure.) A SPR instrument using a dove prism allows a compact and a single axis optical path between an excitation optic fiber and collection optic fibers. This optical configuration simplifies the optical design of SPR instruments without compromise of the analytical performance23(Scheme 1). The light from a white light-emitting diode (LED) source was focused in a 200 μmdiameter optic fiber (excitation optic fiber). Light exiting the excitation fiber optic was collimated using a SMA collimating lens, passed through a spatial filter and a linear polarizer and the dove prism (BK7) with the SPR sensor contacted using

with poly(allylamine hydrochloride) (PAH, Mw ∼ 56k, SigmaAldrich) by employing the widely used carbodiimide coupling reaction.24 A solution containing 20 mM N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC, 99%, Sigma-Aldrich) and 5 mM N-hydroxysuccinimide (NHS, 98%, Fisher Scientific) was injected and stopped for 20 min, followed by 7892

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deionized water flushing. Then, a 1 mg/mL PAH solution at pH 9.2 (adjusted with 1 M NaOH) was injected and allowed to react for 2 h. This procedure yields coating of only the analytical pad, leaving an unmodified bare gold reference pad. Then, this individually functionalized dual-pad SPR sensor can be used for further measurements. Data acquisition was performed at a rate of 1 data point every 6 s with 150 g/mm grating. Each data point was an accumulation of three measurements. Measurement Procedure. The pH responsiveness determined by injection of HCl/NaOH pH standards in ∼0.1 M KCl or different pH buffer solutions (prepared on the basis of the recipes in CRC Handbook of Chemistry and Physics25) into the flow-cell. Solutions were allowed to equilibrate for 3 min prior to collecting spectra. The temperature compensation studies were accomplished by injection of room temperature or cold (8 °C) HCl/NaOH pH standards in 0.1 M KCl, immediately followed by 99 or 900 s SPR measurements. The data acquisition was performed at a rate of 1 data point every 3 s using 1200 g/mm grating with 0.058 nm resolution. For monitoring attachment of a protein, the sensor modification procedure and data acquisition were identical to the one described above; except after EDC/NHS activated the 4-PAD layer, the flow-cell was flushed with several milliliters of phosphate buffered saline (PBS, pH 7.4, MP Biomedicals) and allowed to equilibrate for 15 min. SPR spectra were collected over a 10 min period in PBS, followed by a 30 min exposure to 80 mg/mL bovine serum albumin (BSA, in PBS, SigmaAldrich) and a subsequent 15 min PBS washing step to remove weakly adsorbed species.26

instantaneous. At the 8 min mark, water was injected into the flow-cell. A large SPR wavelength increasing (16.4 nm shift) between two water steps was only observed on the analytical pad with applied voltage pulses. The SPR signal from the reference pad (gray dashed line) only responded to RI changes associated with the injected solutions and returned to the water baseline at the washing step. This demonstrated that only the analytical pad was functionalized with diazonium salt (4-PAD), and the reference pad was not affected. Multiple gold pads may be individually modified with different diazonium salts via serial injection and electrografting (Figure 2). The reference pad was functionalized with 4-

Figure 2. SPR sensorgrams of an individual functionalization dual-pad with two different diazonium salts. Analytical pad (black solid line). Reference pad (gray dashed line). Arrows indicated 2 s, −400 mV pulses applied.

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RESULTS AND DISCUSSION Surface Functionalization of Dual-Pad SPR Sensors. Selective electrografting of diazonium salts of SPR sensing pads is evident in the dual-pad sensorgrams (Figure 1). Because the

nitrobenzenediazonium tetrafluoroborate (4-NDT) first; then, the analytical pad was functionalized with 4-PAD. Upon injection of 4-NDT into the water filled flow-cell, five 2 s, −400 mV pulses were applied on the reference pad, followed by a quick wash with water. Then, the working electrode was exchanged to connect the analytical pad, and the flow cell was subsequently filled with 4-PAD. The same electrografting procedure was applied to the analytical pad. Diazonium salts attached to the gold surfaces were only observed at the single pad having the applied pulses. The surface modification process of the dual-pad SPR system is observed through SPR sensorgrams. (See Figure S-1 in Supporting Information.) Only one gold pad was functionalized with 4-PAD, and the other pad was left unfunctionalized for the following experiments. First, 4-PAD was electrografted onto the analytical pad with five pulses (A). Following a brief water washing step, EDC and NHS (B) were injected into the cell and allowed to react for 20 min. Since the SPR response during (A) showed that the 4-PAD was selectively adhered to the analytical pad, PAH (C) could only bind to the 4-PAD analytical pad. After washing with water (D), the analytical pad (black solid line) presented a 25.8 nm SPR shift relative to the initial water baseline, compared to only 1.8 nm shift on the reference pad (gray dashed line) due to nonspecific binding . The electrografted diazonium salt layer can be employed for protein functionalization as well as attachment of a polymer with EDC/NHS chemistry. When the dual-pad SPR sensor was prepared by electrografting 4-PAD on only one SPR sensing pad, bovine serum albumin covalently bound exclusively to the EDC/NHS activated 4-PAD layer. The observed SPR shift between the two PBS solutions at the analytical pad was a result of covalent BSA attachment, change in the RI of the bulk

Figure 1. SPR sensorgrams of electrografting with diazonium salt (4PAD). Analytical pad (black solid line). Reference pad (gray dashed line). Five arrows indicated five 2 s, −400 mV pulses applied.

two collection fibers are imaged on different areas of the CCD detector, the spectral signature from the two SPR pads are collected and analyzed simultaneously and individually. At time 2.6 min, the 4-PAD solution was injected into the water filled flow-cell and a concomitant red-shift in the resonant wavelength was observed due to the RI increasing from water to 4PAD solution. Then, a series of pulses were applied to only one of the SPR pads (black solid line). Five clear steps demonstrate the electrografting of 4-PAD to that pad. Thirty seconds were allowed between pulses to show that the reaction was nearly 7893

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relationship between the SPR wavelength and RI was employed to estimate the sensitivities of each pad: 2.08 (±0.08) × 103 nm RIU−1 for the PAH-functionalized 4-PAD analytical pad, and 1.93 (±0.06) × 103 nm RIU−1 for the bare gold reference pad. (Note: RIU denotes refractive index unit.) The R2 value for the analytical pad was 0.989 and that for the reference pad was 0.991. The sensitivities have statistically different values (p = 0.98); however, the small difference was considered to be acceptable for the demonstration purposes of this study.28 pH Responsiveness of Dual-Pad Sensing. The analytical pad was coated with a pH sensitive polymer, PAH (pKa of side chain amine groups of PAH is 9.529). An increase in the PAH thickness with increasing pH of the solution has been previous shown.30 At high pH values (e.g., pH 12), the amine group is electrically neutral. A decrease in solution pH leads to ionization of the amine groups; the resulting electrostatic repulsion between the positive charges causes the polymer network to swell and take in water. Hence, the denser PAH at high pH values translates into high surface RI, as opposed to the polymer in the swollen state at low pH values yielding a lower surface RI. These changes, along with confounding bulk RI changes in the aqueous matrix, were monitored by SPR spectroscopy. Analyzing a set of HCl/NaOH pH standards in ∼0.1 M KCl shows both the pH-dependent response of the PAH layer and the ability to improve both accuracy and precision with the dual sensing pads (Figure 4). The result of a monotonically

solution, and nonspecific binding (Figure 3A). The SPR shift between the two PBS solutions at the reference signal reflected

Figure 3. SPR sensorgrams of individually addressed dual-pad SPR sensor exposed to 80 mg/mL BSA. (A) The analytical and reference signals upon introduction of 80 mg/mL BSA at time =10 min for 30 min. Black solid line (analytical pad); gray dashed line (reference pad). (B) After subtracting out the reference signal from the analytical signal, the specific binding of BSA was obtained.

Figure 4. SPR shift vs pH standards in 0.1 M KCl on analytical pad (black solid dots), reference pad (gray solid dots), and the analytical signal minus reference signal (black empty dots). To distinguish, the SPR shift on analytical pad and reference pad started at 0 nm (pH 5), while the analytical signal minus reference signal started at −1 nm (pH 5). The error bars show standard deviation of three individual measurements from different trials.

the change in the RI of the bulk solution and nonspecific binding only. After subtracting out the reference signal from the analytical signal, a 2.9 nm SPR shift accounting for attachment of BSA was obtained (Figure 3B). SPR Calibration. Before any measurements were carried out with the dual-pad SPR sensor system, it was critical to establish that both pads responded identically to the changes in RI or, alternatively, that they could be mutually calibrated to yield identical responses to the same change in RI. Both the PAH-functionalized 4-PAD pad and the bare gold pad demonstrate comparable sensitivities to bulk RI changes. The SPR wavelength was observed at the analytical and reference pads as a function of refractive index for six NaCl standard solutions (the refractive indices were 1.3330, 1.3333, 1.3337, 1.3340, 1.3344, and 1.3347, corresponding to mass concentration of 0.00, 0.20, 0.40, 0.60, 0.80, and 1.00%27). Measurements were in triplicate, and the standard deviation for each data point ranged from 0.02 to 0.23 nm. A linear

increasing SPR shift as the pH increases from the PAH-coated analytical sensor is from the pH-dependent response of the PAH layer and nonspecific influence. The small SPR shifts observed from the reference sensor account for the nonspecific effects. Subtracting the reference signal from the analytical signal leaves only the pH-dependent response of the PAH layer, and yields a bilinear response with a lesser slope at lower pH (5−8) and a greater slope at high pH (9−12.5). While a sigmoidal response curve, with an inflection at pH ∼9.5 was expected, the bilinear response can be justified for the reasons discussed below. An increase in thickness is attributed to a transition in polymer deposition from flat trains to loopy structures as the density of the charges on the polymer chain decreases.31 At low pH, the PAH is fully protonated. As the pH increases to 8, the degree of ionization reaches 70%. As the pH 7894

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the same trend: increasing λSPR with increasing pH for phosphate buffers and relatively blue-shifted λSPR when the borate and carbonate buffers are employed. Subtraction of the reference signal from the analytical signal removes much of the (purported) bulk RI variance assigned to the buffer composition and leaves a λSPR signal correlated with sample pH, not sample composition (Figure 5C). While the improvement in the analytical trend by incorporating the in-channel reference sensor is visually profound, the data highlights a substantial concern with employing the reference sensor. If the reference sensor has nonspecific interactions with the sample that are different than the nonspecific interactions with the analytical sensor, a source of measurement bias will be introduced. By noting the greater drift at each pH across the four runs, the data indicates that the bare gold reference sensor is fouling from the salt matrix at a faster rate than the polymer-coated analytical sensor. In our previous work determining salinity by SPR spectroscopy, the bare gold sensors were conditioned for up to 24 h to stabilize sensor drift in up to 35 ppt KCl solutions.34,35 At each pH, the standard deviation of the analytical minus reference measurements are between the standard deviations of the analytical and reference sensors. Thus, some of this fouling effect is mitigated by referencing, but fouling still accounts for a large source of variance in the data. There is an increase in deviations for measurements made in the cycles following use of the KH2PO4 buffer. A potential explanation involves the binding of phosphate anions to cationic amine groups of PAH, since PAH has been used as a phosphate binder for the treatment of hyperphosphatemia in chronic renal failure.36 Therefore, it is possible that these ionic interactions between chemical species and polymer restrained the pH responsiveness of PAH for two of the cycles while analyzing the pH 9 and 10 standards (Figure 5A); once the solution pH increased past the amine pKa, the phosphate would be released. Temperature Compensation. Temperature fluctuations have limited SPR bioanalyses.14,37,38 A temperature change of 0.1 °C during analysis produces a change in the measured RI on the order of 1 × 10−5 RIU, which is comparable to the detectable signal for an SPR sensor.14 Practical options are to stabilize the temperature of the SPR sensor or compensate for temperature drift. However, as high resolution SPR sensors are developed that approach 10−7 RIU detection limits, temperature stabilization to 0.001 °C is impractical. Consequently, methods for compensation that render measurements effectively temperature-stable to within the detection limit of the sensor platform are needed. Before evaluating the performance of our dual-pad SPR sensor system for compensation for the changes in RI due to temperature changes, it was crucial to know the intrinsic instrumental stability and precision. The standard deviations of continuous 99 s (33 data points) SPR signals at room temperature were calculated. (See Table S-2 in Supporting Information.) In theory, the sample temperature is stable, and no refractive index changes from thermal drift are expected. Predictably, there is no observed trend in improved precision of analysis by incorporating a reference sensor because the system is generally stable. However, subtracting the reference signal resulted in an average instrument limit of detection of 2.4 × 10−6 RIU (0.0048 nm) or 0.024 °C, which was better than the average limit of detection predicted from propagation of errors, when the analytical and reference signals were completely

further increases, PAH continuously loses charges until after pH 12.32 A lesser slope can be expected at pH above 12; irreversible morphological transformation of the pH sensitive polymer has been reported due to the extreme and repeated pH treatment.33 The precision of the analysis improved up to a factor of 10 after incorporating the in-channel reference sensor. (See Table S-1 in Supporting Information.) Both the analytical and reference pads present standard deviation of replicate analysis between 0.1 and 0.2 nm. This standard deviation is a function of intrinsic instrumental precision, any surface fouling that may occur (unlikely in this scenario) as well as variations in bulk refractive index from the water temperature. By mitigating the effect of sample-to-sample changes in bulk RI at the expense of a 2 times increase in the intrinsic instrumental variance, the SPR wavelength standard deviation from analyzing replicate samples was decreased up to 10 times. Incorporation of the reference sensor compensates for bulk matrix density changes. Nine pH standards, prepared from five different buffer recipes, were analyzed. A spread of 2.8 nm in λSPR was observed at the analytical sensor (Figure 5A) and 2.4 nm in λSPR at reference sensor (Figure 5B). Both pads exhibit

Figure 5. SPR vs buffer solutions with pH 6 to 13 on (A) analytical pad and (B) reference pad. (C) After subtracting out the reference signal from the analytical signal. Trial 1 (◊), trial 2 (□), trial 3 (Δ), and trial 4 (○). Different buffers are circled in groups. 7895

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Table 1. Analytical Performance of the Sensors over (A) 0−99 Seconds (12 °C Variation), (B) 600−900 Seconds (0.6 °C Variation), and (C) 0−900 Seconds (15 °C Variation) for pH Standards in 0.1 M KCl (A) pH

analytical SDa (nm)

reference SD (nm)

(anal.-ref.) SD (nm)

RIU LoDb (×106)

compensated to (°C)

improvement times

5 6 7 8 9 10 11 12

0.27 0.23 0.20 0.20 0.27 0.27 0.29 0.29

0.27 0.23 0.21 0.20 0.28 0.25 0.28 0.28

0.0043 0.0037 0.0089 0.0077 0.011 0.013 0.012 0.0057

2.2 1.9 4.5 3.9 5.5 6.7 6.1 2.9

0.022 0.019 0.045 0.039 0.055 0.067 0.061 0.029

63 62 23 26 25 20 24 50

pH

analytical SD (nm)

reference SD (nm)

(anal.-ref.) SD (nm)

RIU LoD (×106)

compensated to (°C)

improvement times

5 6 7 8 9 10 11 12

0.0064 0.0031 0.0025 0.0068 0.0067 0.0026 0.011 0.015

0.0023 0.0034 0.0033 0.0020 0.0022 0.0043 0.0096 0.014

0.0059 0.0051 0.0043 0.0069 0.0073 0.0050 0.0043 0.0034

3.0 2.6 2.2 3.5 3.7 2.5 2.2 1.7

0.030 0.026 0.022 0.035 0.037 0.025 0.022 0.017

1 1 1 1 1 1 3 4

pH

analytical SD (nm)

reference SD (nm)

(anal.-ref.) SD (nm)

RIU LoD (×106)

compensated to (°C)

improvement times

5 6 7 8 9 10 11 12

0.17 0.14 0.12 0.11 0.16 0.16 0.19 0.19

0.15 0.13 0.12 0.11 0.16 0.15 0.18 0.21

0.020 0.021 0.014 0.011 0.018 0.016 0.016 0.018

10.2 10.3 7.0 5.6 9.0 8.0 8.1 8.8

0.10 0.10 0.070 0.056 0.090 0.080 0.081 0.088

8 7 9 10 9 10 12 11

(B)

(C)

a

Standard deviations. bLimit of detection.

uncorrelated, 3.0 × 10−6 RIU (0.0059 nm). Thus, the reference sensor is compensating for some thermal drift, but the level of drift is near the sensor detection limit. The performance metrics of the system (sensitivity of 2.0 × 103 nm RIU−1 and resolution of 0.058 nm when a quadratic curve fit of the SPR spectra about the minima is employed to determine λSPR35) were employed to estimate RIU and temperature stability. Employing two sensing pads within a single channel reduces the effects of temperature drift by up to 60× in these initial demonstrations (Table 1A). In the extreme scenarios, with water temperature changing over 12 °C, subtracting the reference signal reduces the standard deviations in λSPR from 0.27 to 0.0043 nm, resulting in an effective resolution of 2.2 × 10−6 nm or 0.022 °C effective thermal stability. Literature showed a temperature increase of 1 °C produces a decrease of 0.03 in the pKa of PAH,39 and at 10 °C, the pH of water is 7.27 (compared with 7.0 at room temperature). When the pH of KCl solutions approached the pH of water, the polymer experiences a convoluting temperature-dependent pH change. Also, around pKa of PAH, temperature-dependent changes in the pKa of the PAH will mostly influence the degree of polymer protonation and hence the degree of swelling. Therefore, improvements of only 20× were observed between pH 7 and 11. When the temperature was fairly stable (0.6 °C shift) from 600 to 900 s during the measurements, subtracting out the reference signal from the analytical signal resulted in the

average SPR shift being compensated to ±0.0053 nm (±0.026 °C) (Table 1B), which is at our intrinsic instrumental limit of detection. Table 1C comprised the standard deviations over the whole 900 s (15 °C swing), after compensation, the temperature effect was essentially removed (average compensation to ±0.017 nm or ±0.084 °C). The same statistical analysis as Table 1A was done for every 99 s (33 data points) over 891 s for all eight pH values. The precision of the analytical minus reference signal was compared to the precision of the analytical signal, and the propagation of errors predicted precision at the 95% confidence level. Relative to the uncompensated analytical signal, 34 out of 72 calculated results were significantly better; 30 out of 72 were with no improvement, and 8 out of 72 were with negative improvement. Comparing the standard deviations after compensation with propagation errors, 58 out of 72 calculated results were significantly better, 10 out of 72 were with no improvement, and 4 out of 72 were with negative improvement. This meant incorporation of the reference sensor compensated some uncertainties but not all of them, for reasons discussed above. Figure 6 showed the SPR shift before and after reference pad compensation over three periods of time: the whole 900 s (15 °C variation), first 99 s (12 °C variation), and 600−900 s (0.6 °C variation). Blue-shifts on both the analytical and reference pads due to the RI decreasing, resulting from warming of the solution were observed. However, after subtracting out the 7896

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ACKNOWLEDGMENTS

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REFERENCES

Article

Funding from the National Institutes of Health is acknowledged through grant no. R01EB0044761.

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Figure 6. SPR shift vs time showing reference pad compensation for temperature changes using pH = 12, 0.1 M KCl solution for 0−900 s (15 °C variation). Inset: SPR shift vs time for the first 0−99 s (12 °C variation) and last 600−900 s (0.6 °C variation). Black solid line (analytical pad), gray dashed line (reference pad), and black dotted line (analytical signal minus reference signal).

reference signal, the shift was nearly entirely accounted for in all three representative periods.

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CONCLUSIONS In this paper, we demonstrated that a dual-pad SPR sensor was successfully and individually functionalized with two diazonium salts on two gold pads in the same fluidic channel. We were able to demonstrate both analytical pad and reference pad responded identically to the changes in refractive index or alternatively. The performance of electrografting 4-PAD as a linker for biomolecular detection was demonstrated by measuring the specific binding of BSA using the dual-pad SPR sensor. Moreover, when PAH coated to the analytical pad, after subtracting out the reference signal from the analytical signal, we were able to show the SPR signals response monotonically to the pH effect only, also realizing compensation of the apparent temperature to the second decimal over 15 °C swing. Therefore, the reference pad in the same channel allowed us to subtract out all nonspecific effects (e.g., bulk refractive index changes and temperature variations), leaving only the specific binding of the analyte of interest. This reference pad not only improved the robustness of SPR measurements by compensating for background bulk refractive index variations but also helped eliminate potential false positive responses (e.g., from nonspecific binding). We believed that our work here would provide a new possibility for high sensitivity and selectivity SPR measurement of chemical or biomolecular interactions.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +1 (302) 831-2561. Fax: +1 (302) 831-6335. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 7897

dx.doi.org/10.1021/ac301528z | Anal. Chem. 2012, 84, 7891−7898

Analytical Chemistry

Article

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dx.doi.org/10.1021/ac301528z | Anal. Chem. 2012, 84, 7891−7898