Plastic-based lateral flow immunoassay device for electrochemical detection of NT-proBNP

Abstract

Here we report an easily fabricated, plastic-based lateral flow device for carrying out metalloimmunoassays. The device is called ocFlow to emphasize the open-channel design. We have shown that the ocFlow is capable of magnetic microbead (MµB)-based metalloimmunoassays for the detection of two types of immunoconjugates: a model composite (MC) and a sandwich immunoassay for the heart failure marker NT-proBNP. In both assays, Ag nanoparticles (AgNPs) were used as electrochemically detectable labels. NT-proBNP and MC concentrations as low as 750.0 pM and 10.0 pM, respectively, could be detected using the ocFlow device. Four key conclusions can be drawn from the results presented herein. First, immunoconjugates attached to the MµBs can be transported in the flow channel using combined hydrodynamic and capillary pressure passive pumping. Second, the ocFlow device is capable of on-chip storage, resolvation, and conjugate formation of both the MC and NT-proBNP composites. Third, electrochemical detection can be conducted on analytes suspended in serum by rinsing the electrodes with a wash buffer. Finally, and perhaps most significantly, the assay is quantitative and has a detection limit for NT-proBNP in the high picomolar range when the necessary reagents are stored on the device in a dry form.

Graphical Abstract

A low-cost plastic-based lateral flow device was developed to detect NT-proBNP, a heart failure marker, using the electrochemical metalloimmunoassay technique.

Introduction

Disease biomarkers are often present in trace amounts in biological samples, and therefore they require ultrasensitive methods for detection and quantification. Metalloimmunoassays employ metal-labeled antibodies for detection, and in some cases, they have been shown to provide appropriately low limits of detection.^1–3

We have been developing an electrochemical metalloimmunoassay for the heart failure biomarker N-terminal prohormone brain natriuretic peptide (NT-proBNP), which is a short peptide.^4–7 The baseline concentration of NT-proBNP in the blood of a patient depends on a number of variables, including their stage of heart failure, comorbidities, age, and race.^8–12 Changes in the baseline level of NT-proBNP may be an indication of disease progression, and therefore there is a need for monitoring NT-proBNP levels at home.^{13, 14}

Point-of-care (PoC) diagnostic devices have become increasingly available because of their ability to provide fast (typically <10 min) diagnostic testing in non-laboratory environments.^15–17 Among these devices, lateral flow immunoassays (LFIA) have been successfully applied for both qualitative and quantitative detection of biomarkers. One of the most well-known such devices is the home-use pregnancy test,^18–21 although the recent introduction of home-use LFIAs for the SARS-CoV-2 virus has also underscored their utility.^{22, 23} LFIAs are typically paper-based devices in which the sample flows from one end of a paper strip to the other, picking up predried assay reagents (usually antibodies) en route. The analyte of interest forms an immunoconjugate with the dried antibodies and a colorimetric signal is typically observed.¹⁸

The electrochemical metalloimmunoassay for NT-proBNP developed in our lab is essentially an antibody sandwich immunoassay requiring two reagents. One reagent is a capture antibody (SAb) immobilized on a magnetic microbead (MµB-SAb). The other is a detection antibody attached to an Ag nanoparticle (AgNP-Ab).²⁴ These two reagents act in concert to provide a picomolar limit of detection for NT-proBNP.^{4, 6} Specifically, the MµBs preconcentrate the assay conjugate over a working electrode (WE), which itself is configured over a magnet.^{4, 25, 26} The AgNPs provide a maximum amplification factor of 250,000 upon oxidation.^{5, 27}

Following preconcentration of the immunoassay over the WE, a series of electrochemical steps (discussed later) are carried out to quantitatively detect the AgNP labels, which are correlated to the NT-proBNP concentration in the sample.^{4, 6} One challenge we have encountered with this approach is that the immunoconjugates attached to the MµBs become trapped in the porous matrix of the paper device, and therefore they do not properly move down the paper strip.^{28, 29} To solve this problem, we and others developed hollow channel paper-based devices wherein the sample flow occurs in the hollow microfluidic channel instead of the porous matrix.^{30, 31} Even in these types of devices, however, transport remains a problem.

Ultimately, we developed a hybrid paper and 3D-printed lateral flow device (called hyFlow) that was successful in preventing the retention of MµBs.³² We also showed that both MµBs and AgNPs could be stored/dried in the device and that MµB-AgNP conjugates could be formed in situ within the flow channel. The assembly of this device was cumbersome, however, and it required non-disposable 3D-printed parts. To resolve these and other problems, we now report a type of surface flow device (SFD).^33–35

SFDs are microfluidic devices in which fluidic pathways are defined by patterning hydrophilic regions on an open hydrophobic surface. Passive pumping in SFDs can be implemented by Laplace pressure,^{33, 36, 37} hydrodynamic pressure,³⁸ and capillary forces.³⁹ Accordingly, reagents and analytes can be transported without an external power requirement. Other advantages of open fluidic pathways in SFDs are minimal non-specific adsorption, direct environmental accessibility, clear optical path, and reduced clogging.³³ Therefore, SFDs have been used for many applications such as automated cell culture,⁴⁰ pathogen detection,⁴¹ nanoparticle synthesis,³⁷ and for carrying out chemical reactions on the micro-scale.⁴²

In the present study, we have developed a novel, low-cost method for fabricating an electrochemical SFD. Because this is an open-channel device, we henceforth refer to it as ocFlow. We show that the ocFlow device can be used to detect two different types of immunoconjugates: a model composite (MC) and an NT-proBNP immunosandwich (Figure S1, Electronic Supplementary Information, ESI). On the basis of our findings, we conclude that the ocFlow has four significant attributes. First, the device is low-cost and disposable. Second, it provides effective transport of MµBs. Third, reagents necessary for the electrochemical metalloimmunoassay (e.g., AgNP, MµBs, and antibodies) can be stored on the device and the immunoreaction can be conducted in situ. Fourth, analytes suspended in complex biological media, such as serum, can be detected.

Experimental Section

Chemicals and materials.

All solutions were prepared using deionized (DI) water (>18.0 MΩ-cm, Milli-Q Gradient System, Millipore, Burlington, MA). Phosphate buffered saline (PBS) pH 7.4 (P3813), superblock blocking buffer containing PBS (SBB) (cat. no. 37515), siliconized low-retention microcentrifuge tubes, HAuCl₄, and KNO₃, were purchased from Fisher Scientific (Pittsburgh, PA). All PBS concentrations were 1X. The 5 kDa mPEG-SH was obtained from Nanocs (New York, NY). Sucrose, D-(+)-trehalose dihydrate, and normal human serum were purchased from Millipore-Sigma (Burlington, MA).

Citrate-capped AgNPs (nominal diameter = 20 nm) were purchased from nanoComposix (San Diego, CA). NT-proBNP, monoclonal immunoglobulin G anti-NT-proBNP 13G12cc detection Abs, and nonbiotinylated monoclonal immunoglobulin G anti-NT-proBNP 15C4cc capture Abs were obtained from HyTest (Turku, Finland). Amicon Ultra 0.5 mL centrifugal filters (10 K) were purchased from Millipore-Sigma (Taunton, MA). Conductive carbon paste (Cl-2042) was purchased from Engineered Conductive Materials (Delaware, OH). Cylindrical neodymium magnets (1/16 in. × 1/2 in., N48) were purchased from Apex Magnets (Petersburg, WV). Transparency sheets from VU-Color (Cat. NO. PP6901), self-adhesive laminating sheets (Cat. No. 461253) from Avery, and superhydrophobic spray from NeverWet (ASIN: B01M4KO4EP) were purchased from Amazon. All chemicals and reagents were used without further purification unless otherwise specified.

Instrumentation.

Information about instrumentation is available in the ESI.

Electrochemistry.

Electrochemical measurements were performed using a CH Instruments model 760B electrochemical workstation (Austin, TX). Electrochemical measurements on the ocFlow device were performed to detect AgNP labels using a previously published protocol.^{4, 5, 25, 26} Briefly, the potential of the WE was stepped from 0.0 to 0.8 V (all potentials are vs. a carbon quasi-reference electrode (CQRE)) for 12.0 s to oxidize pre-deposited Au⁰ to Au³⁺ to initiate the galvanic exchange (GE) process. Ag⁺ formed during GE was then electrodeposited onto the WE by stepping the potential from 0 to −0.7 V for 50.0 s. These two steps were repeated a second time. Next, linear scan voltammetry was applied wherein the potential was swept from −0.7 to 0.2 V at a scan rate of 50 mV/s. Finally, square-wave anodic stripping voltammetry (SWASV) was applied wherein the potential was again swept from −0.7 to 0.2 V to oxidize Ag from the WE. The square-wave frequency, amplitude, and voltage increments of 100.0 Hz, 50.0 mV, and 4.0 mV, respectively were used for this scan.⁴³ The peak currents for the SWASV voltammograms were determined and correlated to the concentration of the target.

Device fabrication.

The procedure for fabricating the ocFlow device is presented in ESI (Figure S2). Briefly, however, a hydrophilic transparency sheet was printed with hydrophobic ink using a laser printer to define a stopper and a sample drying zone. Second, the electrochemical detection zone was defined by stencil printing carbon-paste electrodes over the transparency sheet. Third, the sample flow areas (i.e., the channels, detection zone, and inlet) were defined by bonding a superhydrophobic self-adhesive sheet with the transparency sheet. Finally, the device was cut at the edges and bonded with porous paper at the end to yield the final device.

Formation of metalloimmunoassays for different assay types.

Preparation of the AgNP-Ab, MµB-SAb, and MµB-15C4cc conjugates are provided in the ESI. A total of four different types of assays were conducted on two assay composites. The assays are categorized according to the way the sample and the reagents are introduced at the inlet of the ocFlow. For the ‘wet’ assay, the MC is made off-chip and introduced onto the device in the form of a suspension. The MC assay was formed by conjugating MµB-SAb and AgNP-Ab via an interaction between the two Abs: 13G12cc and SAb (Figure S1). Specifically, 16.0 µL of the as-prepared MµB-SAb were added to 100.0 µL of the desired concentration of AgNP-Ab and then incubated for 30.0 min in the tube revolver at 30 rpm. The MC was then washed with 1% BSA w/v in PBS solution five times using magnetic separation wherein the MµBs were collected on the wall of a microcentrifuge tube with a neodymium magnet, the supernatant was removed, and the conjugate was resuspended in PBS and washed again. Subsequently, the MC was resuspended in 16.0 µL of PBS. 2.0 µL of this assay was combined with 18.0 µL of PBS or 100% serum to yield a final sample volume of 20.0 µL. This solution was then introduced at the inlet of the ocFlow device to be transported to the detection zone for electrochemical analysis.

For the ‘dry’ assay, a procedure identical to the ‘wet’ assay was used to prepare the MC. However, after washing with the BSA solution, the MC was resuspended in 16.0 µL of 50.0 wt% sugar solution instead of PBS. The 50.0 wt% sugar solution was prepared by adding an appropriate amount of 1:1 mixtures of sucrose and D-(+)-trehalose dihydrate in DI water. 2.0 µL of this mixture (MC in sugar solution) was dried overnight onto the drying zone of the ocFlow device. The next day, 20.0 µL of PBS solution was used to resuspend the pre-dried MC. Subsequently, the suspended MC was transported to the detection zone for electrochemical analysis.

For the ‘instant mix-1’ assay, the MµB-SAb conjugate was prepared following the procedure described in the ESI. However, the as-prepared MµB-SAb conjugate was resuspended in 100.0 µL of sugar solution instead of a 1% w/v BSA in PBS. Subsequently, 2.0 µL of MµB-SAb in the sugar solution was dried overnight onto the drying zone of the device. The next day, 20.0 µL of the desired concentration of AgNP-Ab in SBB was used to resuspend the dried MµB-SAb, followed by ~5 min of incubation time for on-chip MC formation. The as-formed MC was then transported to the detection zone for electrochemical analysis.

For NT-proBNP detection, an ‘instant mix-2’ assay was conducted. Specifically, 2.0 µL of MµB-15C4cc in sugar solution was pipetted onto the drying zone followed by the addition of 2.0 µL of excess AgNP-Ab solution (~7500 pM) on top of the MµB-15C4cc droplet. The mixture was dried overnight and resuspended the next day using a 20.0 µL of desired concentration of NT-proBNP solution in SBB. The NT-proBNP assay conjugate (Figure S1) was formed at the inlet for ~5 min after which it was transported to the detection zone for electrochemical analysis. The exact procedure for reagent resuspension and immunoconjugate transport to the detection zone is discussed in the Result and Discussion section.

Result and Discussion

Description of the GE/ASV process.

The method for detecting NT-proBNP is based on a metalloimmunoassay comprising MµB-15C4cc and AgNP-Ab (Ab = 13G12cc). NT-proBNP is sandwiched between these two reagents to yield the assay conjugate: MµB-NT-AgNP (Scheme 1). The solution containing MµB-NT-AgNP is then transferred to the WE for electrochemical analysis. Electrochemical detection of the assay is conducted in several steps as described in detail in the Experimental Section and summarized by Scheme 1. First, the Au, pre-deposited onto the WE, is electrochemically oxidized to form Au³⁺. Second, Au³⁺ diffuses to the AgNPs localized on the electrode via the magnetic force. This initiates a process known as galvanic exchange (GE).²⁶ As shown in eq 1, the GE process takes advantage of the difference in the standard potentials of two metals such that the less noble Ag (E⁰ = 0.79 V) spontaneously exchanges with the more noble Au³⁺ (E⁰ = 1.52 V) to yield Au and Ag⁺.

Scheme 1.

Schematic representation of NT-proBNP detection using the galvanic exchange/anodic stripping voltammetry (GE/ASV) technique.

$$3A{g}_{\left(s\right)}^o+A{u}_{\left(aq\right)}^{3+}\to A{u}_{\left(s\right)}^o+3A{g}_{\left(aq\right)}^{+} \Delta E=0.7V$$ (1)

Third, the Ag⁺ resulting from GE is electrodeposited onto the WE as Ag. Finally, the electrochemical technique of square wave anodic stripping voltammetry (SWASV) is applied to oxidize Ag from the WE.⁴⁴ The Ag oxidation current from the SWASV step is then correlated with the concentration of NT-proBNP in the sample.

Description of the device.

As discussed in the Experimental Section and ESI, we have developed an SFD using contrasting hydrophilic and hydrophobic patterns in an open-channel configuration. The ocFlow device features a sample inlet with a hydrophobic reagent-drying zone, a flow channel with a stopper, an electrochemical detection zone, and a paper sink (Scheme 2).

Scheme 2.

Schematic diagram of the ocFlow device.

The flow channel and electrochemical detection zone were defined by bonding a hydrophobic, laser-patterned adhesive sheet with a hydrophilic transparency sheet. For on-chip storage and drying of reagents, a hydrophobic circle was printed at the inlet of the device using a laser printer. The drying zone was made hydrophobic to facilitate the resuspension of the dried MµBs during the operation of the device. In the absence of this hydrophobic region, the MµBs tended to adhere strongly to the hydrophilic transparency sheet. Note that the edges of the inlet were left hydrophilic to facilitate sample loading (Scheme 2).

A hydrophobic stopper line was also printed in the channel to stop the sample from flowing into the channel prematurely during in-situ assay formation (see Movie S1). Three carbon paste electrodes were screen printed on the transparency sheet for electrochemical detection. Finally, a piece of chromatography paper is present at the end of the device to absorb waste liquid and drive the liquid forward in the flow channel. The shape of the paper sink was kept triangular to ensure the compactness of the device. In general, any appropriate shape of paper can be applied to absorb the waste liquid. Once constructed, the device was placed over a platform with a small magnet positioned beneath the WE to capture immunoconjugates attached to the MµBs prior to electrochemical analysis.

Spontaneous fluid flow arises from a combination of hydrodynamic and capillary pressure. When a sample droplet is pipetted onto the inlet, hydrodynamic pressure is generated due to a rise in liquid height causing the liquid to move along the channel. This excess pressure can be increased by increasing the droplet volume (height) at the inlet. Additionally, capillary pressure is also generated at the liquid front due to favorable free energy caused by spreading of the liquid over the hydrophilic surface.

As discussed in the Introduction, MµBs were entrapped in the cellulose matrix in our previously reported paper-based microfluidic channels thereby reducing detection efficiency. Here, we hypothesized that because the ocFlow device eliminates the paper matrix, MµB transport would be enhanced.

Types of assays.

As mentioned previously, both the MC and the NT-proBNP composites were analyzed on the ocFlow device. Additionally, four different types of assays were conducted on these two composites (Scheme 3, also see the Experimental Section for the details). Each of these will be discussed separately.

Scheme 3.

Schematic representation of four assay types investigated using the ocFlow device.

Wet assay in buffer.

The ‘wet’ assay was conducted to study the effect of MµBs transport on assay performance. As discussed in the Experimental Section, the ‘wet’ assay is performed by preparing the MC off-chip in PBS and then introducing it directly into the device inlet. For these experiments, the AgNP concentration was 100.0 pM. We call this suspension Wet/PBS.

After preparing the Wet/PBS suspension, the following solutions were pipetted sequentially into the inlet of the device: 100.0 µL of PBS to pre-wet the channel and electrode, 20.0 µL of the Wet/PBS sample, and then five sequential 100.0 µL rinse volumes of PBS. When the final rinse buffer ceased to flow (due to saturation of the paper reservoir), the electrochemical analysis was performed. Note that because the ‘wet’ assay does not require reagent drying and on-chip conjugate formation, the hydrophobic ink that defines the drying zone and the stopper was not printed on the device.

As discussed earlier, electrochemical detection of the assay proceeds through several steps, ultimately involving oxidation of the AgNP labels and subsequent detection of the quantity of Ag⁺ released by SWASV. Figure 1 shows the final SWASV voltammograms for Wet/PBS ([AgNP] = 100.0 pM) obtained using five independently fabricated ocFlow devices. Qualitatively, the Ag oxidation current peaks in the voltammograms indicate reproducible transport of the MC (MµB-AgNP) to the electrochemical detection zone. Quantitatively, the average peak height of the five measurements is 228 µA (coefficient of variation, COV = ~11%). These results are comparable to those we previously determined using screen-printed carbon paste electrodes (~260 µA, COV = ~10%). In that case, however, the MC was placed directly onto the WE so that MµBs loss by entrapment was not possible. Because the results of the two sets of experiments are so similar, we conclude that most of the MC introduced at the inlet of the ocFLow device flows efficiently through the channel and reaches the detection zone. This also shows that there is minimal non-specific binding of reagents onto the surface of the ocFlow device. This finding is important for studies discussed later in which the assay is formed from predried reagents.

Figure 1.

Baseline-corrected SWASVs for the ‘wet’ assay. The five nominally identical experiments were carried out using independently fabricated ocFlow devices and the MC ([AgNP] = 100.0 pM) suspended in PBS. The position of the SWASV peaks varies because of the use of a CQRE.

Wet assay using serum samples.

In this section, we demonstrate the performance of the ocFlow device for analyzing immunoconjugates suspended in serum samples. This part of the study is important because electrode biofouling is a common problem with electroanalytical assays carried out using biological samples.^{45, 46} One advantage of an open-channel, lateral-flow device is that the electrodes can be quickly rinsed by flowing a volume of buffer over the electrodes. Accordingly, we investigated the effect of washing on the detection of the MC suspended in a serum sample.

Just as in the previous section, this ‘wet’ assay was conducted using the MC having an AgNP concentration of 100.0 pM. The MC was prepared off-chip in PBS. The as-formed MC was then separated from the buffer solution using a magnet and resuspended in 100% human serum. We call this suspension Wet/serum. The assay was carried out by pipetting the following solutions into the inlet of the device: 100.0 µL of PBS to pre-wet the electrode, 20.0 µL of the Wet/serum sample, and then different numbers of 100.0 µL aliquots of PBS. When the final wash buffer in each experiment ceased to flow, the electrochemical analysis was performed.

Figure 2a shows representative SWASV voltammograms for Wet/serum ([AgNP] = 100.0 pM) when different numbers of wash volumes were used for rinsing the electrode. Qualitatively, these voltammograms indicate that the Ag oxidation current increases as a function of the number of rinse steps employed, indicating the progressive removal of passivating biomolecules from the surface of the electrode. Note that without any rinsing, complete suppression of the electrochemical signal is observed.

Figure 2.

(a) Representative baseline-corrected SWASVs for the ‘wet’ assay. The experiments were carried out using independently fabricated ocFlow devices and the MC ([AgNP] = 100.0 pM) suspended in 100% human serum. The number of rinse steps (100.0 µL of PBS per rinse step) for each assay is indicated in the legend. The position of the SWASV peaks varies because of the use of a CQRE. (b) Boxplot obtained by measuring the peak height of the voltammograms like those shown in (a). Each box was obtained using data from five independently fabricated ocFlow devices. The result of the control experiment where the MC was suspended in PBS (Wet/PBS) instead of serum is also included.

For quantitative comparison of the foregoing results, a boxplot is shown in Figure 2b. It reveals how the peak height of voltammograms like those in Figure 2a change as the number of 100 µL wash volumes increases. In this plot, each box represents measurements from five independently fabricated ocFLow devices. Measurements from the control experiment (Wet/PBS, [AgNP] = 100.0 pM) are also shown as the last set of data in the box plot. These results indicate that experiments carried out using less than five wash volumes yielded significantly lower currents and higher COVs than the control experiment (no serum). The results from the experiment with five wash steps were compared with the Wet/PBS experiment by performing a two-tailed Student’s t-test (α = 0.05). The p-value of 0.15 indicates no statistical difference between these two results, demonstrating that biofouling of the electrode is eliminated after five wash steps.

To further underscore the importance of multiple rinsing steps, the MC ([AgNP] = 100.0 pM) suspended in 100% serum was analyzed by performing a similar ‘wet’ assay experiment as discussed above, but with a slightly different washing procedure. Specifically, instead of rinsing five times, each with 100.0 µL of PBS (total of 500.0 µL of PBS), a single 500.0 µL PBS rinse volume was used. The results of these experiments for five independently fabricated ocFlow devices are shown in Figure S4 in the ESI. The average peak height and standard deviation for these voltammograms was 52 ± 52 µA, indicating both poor signal quality and poor reproducibility. We conclude that electrode cleaning is more effective when the washing procedure is performed in multiple steps. The key point from this part of the study, however, is that appropriate electrode washing procedures yield high-quality data even for serum samples.

Effect of resuspending predeposited conjugate on assay performance.

In a typical LFIA device, the immunoconjugate is formed on-chip. This is achieved by immobilizing two different antibodies in the flow channel of the device, and then resuspending one of them during sample flow. This functionality allows the operation of the device in a non-laboratory environment, which is an important property of a PoC device.

Using a paper-based device, we previously showed that pre-drying of reagents resulted in a loss of ~60% of the analytical signal compared to assays in which the immunocomplex was formed in solution prior to injection into the device.³² We attributed this loss of assay performance mainly to passive resuspension of the dried conjugate. The open-channel design reported here makes it possible to use an active technique for resuspending the conjugate. Specifically, the pipette used to introduce the sample is also used to facilitate complex formation (See Movie S1). We next compare the relative effectiveness of active and passive resuspensions on assay performance.

For this analysis, we conducted an ‘instant mix-1’ assay using the MC (Scheme 3). As discussed in the Experimental Section, the immunoconjugate is formed on-chip in the ‘instant mix-1’ assay. Specifically, a 2.0 µL aliquot of MµB-SAb in a sugar solution was dried overnight onto the drying zone of the device (Scheme 3, see Experimental Section for details). The sugar solution is used to inhibit protein denaturation during and after drying.^{47, 48} Unless stated otherwise, all the reagents in this study were dried in the presence of a sugar matrix.

The dried MµB-SAb was resuspended by placing 20.0 µL of AgNP-Ab in SBB solution ([AgNP] = 100.0 pM) into the device inlet followed by active resuspension (mixing of dried reagents with the sample solution by pumping with a pipette for ~10 s). The hydrophobic barrier created by the stopper (Scheme 2) restricts the flow of the reaction mixture into the channel during active resuspension and a subsequent 5 min incubation period (Movie S1). Next, an additional 100.0 µL of PBS was added to the reaction mixture to drive it past the stopper barrier and toward the detection zone where the MC is captured by a magnet present beneath the WE. Five additional 100.0 µL rinsing volumes of PBS were added to remove unconjugated reagents from the vicinity of the WE. After the final rinse volume, the electrochemical analysis was performed.

An identical experiment, but using passive resuspension of MµB-SAb was also conducted for comparison to the active mixing method. For passive mixing, 20.0 µL of the AgNP-Ab solution ([AgNP] = 100.0 pM) was simply left in contact with the predried MµB-SAb at the inlet for 5 min. Next, the contents of the inlet were transported to the WE for the electrochemical analysis as discussed in the previous paragraph.

The average SWASV peak heights obtained from the foregoing experiments are shown in Figure 3. The results indicate that active resuspension yields an average peak height that is about three times higher than the passive resuspension method. Figure 3 also compares the active resuspension method to the ‘wet’ assay (Wet/PBS) discussed in the previous section. A two-tailed Student’s t-test (α = 0.05) yields a p-value of 0.30, indicating no statistical difference in the assay performances of the ‘wet’ and ‘instant mix-1’ assays when active resuspension is used. The poorer performance of the passive resuspension method may be attributed to the less effective resuspension of MµB-SAb. Some possible reasons for this are discussed in the ESI in the context of Figure S5.

Figure 3.

Histogram showing the effect of passive and active resuspension on the analysis of the MC ([AgNP] = 100.0 pM) for the ‘instant mix-1’ assay. A control experiment (i.e., Wet/PBS) is also shown. The error bars represent the standard deviation from the mean for 3–4 independently fabricated ocFlow devices.

On the basis of the results reported in this section, we conclude the following. First, the MµB-SAb reagent can be dried and stabilized on the device in the presence of a sugar solution. Second, the current signal obtained using active resuspension is, within error, identical to that of the ‘wet’ assay. This indicates that the MC can be formed on-chip without loss of assay performance. While the magnitude of the electrochemical response is suppressed by a factor of three in the case of passive mixing, this simpler method is still viable and results in a low run-to-run COV.

Calibration curve for MC detection using the ‘instant-mix 1’ assay.

Up to this point, all analyses were carried out using the MC with a single AgNP concentration (100.0 pM). However, the primary purpose of a diagnostic sensor is to measure different concentrations of biomarkers. Therefore, in this section, we provide a dose-response curve by analyzing the MC as a function of the AgNP concentrations (0–200.0 pM) using the same ‘instant-mix 1’ assay discussed in the previous section.

Representative voltammograms for this set of experiments are shown in Figure 4a. Qualitatively, these voltammograms indicate that the Ag oxidation current increases as a function of the AgNP concentration in the samples. The calibration curve in Figure 4b was generated by plotting the peak heights of voltammograms like those in Figure 4a against the AgNP concentration. The data indicate that the current signal increases monotonically and non-linearly as a function of the AgNP concentration. Note that the signal begins to plateau at concentrations higher than ~150.0 pM AgNPs. The signals from samples containing less than 10.0 pM of AgNPs could not be differentiated from the blank, indicating that the dynamic range of the assay is 10.0–150.0 pM. Another important aspect of Figure 4b is that the average COV of the data is <11%.

Figure 4.

(a) Representative baseline-corrected SWASV voltammograms for the ‘instant mix-1’ assay conducted on MC samples having concentrations in the range of 0–200.0 pM AgNPs. (b) Calibration curve obtained by measuring the peak height of voltammograms like those shown in (a). An exponential function (dashed curve) was used to fit the calibration data points. Error bars represent the standard deviations from the mean for 3–4 measurements obtained using independently fabricated ocFlow devices. The position of the SWASV peaks varies because of the use of a CQRE.

An exponential function (Equation S1, ESI) was used to fit the calibration data (dashed line). The fit parameters of the curve are provided in Table S1 in the ESI.

Calibration curve for the NT-proBNP assay.

Following the rather simplistic MC assay, we turned our attention to the detection of NT-proBNP, which is the primary focus of our sensor-related studies. An ‘instant mix-2’ assay was conducted for this analysis (Scheme 3). For this assay, 2.0 µL of MµB-15C4cc solution was pipetted onto the drying zone followed immediately by 2.0 µL of the AgNP-Ab solution ([AgNP] = ~7500 pM). Both reagents were dried overnight in the presence of the sugar solution. Subsequently, 20.0 µL of an NT-proBNP solution in SBB was pipetted into the device. Finally, a procedure identical to that of the ‘instant mix-1’ assay was carried out for resuspension of the dried reagents, conjugate transport, and electrochemical analysis.

Representative voltammograms for this set of experiments are shown in Figure 5a. The calibration curve in Figure 5b was generated by plotting the peak heights of voltammograms like those in Figure 5a against the NT-proBNP concentration. Unlike the MC analysis, the current signal in this case is a linear function of the NT-proBNP concentration (fitting details are provided in Table S2). From the plot, we estimate that the dynamic range of this assay is 750–2500 pM. The average COV of the combined data set is ~15%.

Figure 5.

(a) Representative baseline-corrected SWASV voltammograms for the ‘instant mix-2’ assay conducted using NT-proBNP samples in the concentration range 0–3840 pM. (b) Calibration curve obtained by measuring the peak height of voltammograms like those shown in (a). A linear function (dashed line) was used to fit the calibration data points. The error bars represent the standard deviation from the mean for 3–4 measurements obtained using independently fabricated ocFlow devices. The position of the SWASV peaks varies because of the use of a CQRE.

These results indicate that both MµB-15C4cc and AgNP-Ab are stable, at least overnight, when dried in the presence of a sugar solution. Moreover, upon hydration the full NT-proBNP conjugate can be formed on-chip, starting from dry assay components.

Analysis of NT-proBNP in serum samples.

In this section, we simulated a more realistic PoC scenario wherein a human serum sample containing NT-proBNP is directly analyzed using the ocFlow device without any off-chip conjugate preparation requirements. Accordingly, we conducted an identical ‘instant mix-2’ assay, as discussed in the previous section, with the exception that NT-proBNP was suspended in a 100% human serum sample instead of SBB. The concentration of NT-proBNP in these experiments was kept constant at 3840 pM.

Figure 6 shows the SWASV voltammograms for this set of experiments obtained using four independently fabricated ocFlow devices. The average peak height for the four measurements was 164 ± 27 µA. These results are statistically similar (t-test, α = 0.05) to the results obtained in the previous section when the NT-proBNP (3840 pM) was suspended in SBB solution (Figure 5b).

Figure 6.

Representative baseline-corrected SWASVs for the ‘instant mix-2’ assay. These four nominally identical experiments were carried out using independently fabricated ocFlow devices. The NT-proBNP (3840 pM) was suspended in 100% human serum. Five rinse steps (100.0 µL of PBS per rinse step) were applied for each experiment. The position of the SWASV peaks varies because of the use of a CQRE.

These results indicate two important points. First, just as the MC, the NT-proBNP assay can also be conducted in the serum sample with subsequent rinsing of the electrode. Second, and more importantly, the assay is highly specific, i.e., the components of serum matrix do not interfere with the NT-proBNP capture and detection efficiency.

Summary and Conclusions

In this study, we have reported the development of a novel plastic-based lateral flow immunoassay device for detecting a model reagent (the MC) and NT-proBNP, a biomarker for heart failure. This device was developed to overcome some of the problems associated with MµBs transport, reagent resolvation, and biofouling in paper devices.^{4, 25, 30, 32}

Five key conclusions can be drawn from the results presented herein. First, immunoconjugates attached to the MµBs can be transported in the flow channel using combined hydrodynamic and capillary pressure passive pumping. Second, the ocFlow device is capable of on-chip storage, resolvation, and conjugate formation of both the MC and NT-proBNP assay composites. Third, proper mixing and resuspension (i.e., active resuspension) of dried reagents is necessary to ensure optimal assay performance. Fourth, electrochemical detection can be conducted on analytes suspended in serum by rinsing the electrodes with a wash buffer. Finally, and perhaps most significantly, the assay is quantitative and has a detection limit for NT-proBNP in the high picomolar range when the necessary reagents are stored on the device in a dry form.

Currently, we are working on optimizing the NT-proBNP assay so that it operates in the risk stratification range for heart failure of 53 pM to 590 pM NT-proBNP.^{9, 10} The next step is to use the ocFlow device to detect NT-proBNP in patient serum samples. The results of these experiments will be reported in due course.

One final point merits mention. Although the focus of this study is NT-proBNP detection, the ocFlow device could be appropriate for any assay requiring on-chip assay formation, efficient micron-size particle transport, and washing. Given the low-cost and easy-to-fabricate aspect of the device, it seems likely that this SFD could be easily developed for other targets.

Supplementary Material

supporting info

Configuration of NT-proBNP assay composite and the model composite (Figure S1); Fabrication of the ocFlow device (Figure S2); Image of the ocFlow device (Figure S3); Voltammograms for the Wet/serum experiment with a single 500.0 µL wash step (Figure S4); Effect of passive resuspension on the assay performance (Figure S5); Fit equation and parameters for MC calibration curve (Table S1); Fit equation and parameters for NT-proBNP assay calibration curve (Table S2); Additional experimental methods describing instruments used for the experiments, and AgNP-Ab and MµB-SAb conjugate preparation.