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A lateral flow aptasensor for small molecule targets exploiting adsorption and desorption interactions on gold nanoparticles Omar A Alsager, Shalen Kumar, and Justin M Hodgkiss Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017

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Analytical Chemistry

A lateral flow aptasensor for small molecule targets exploiting adsorption and desorption interactions on gold nanoparticles Omar A. Alsager a,b,c, Shalen Kumard,e, and Justin M. Hodgkiss b,c * a

King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia

b

School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington 6012, New Zealand

c

The MacDiarmid Institute for Advanced Materials and Nanotechnology, New Zealand

d

School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand

e

AuramerBio Ltd, Callaghan Innovation Quarter, 69 Seaview Road, GraceFiled, Lower Hutt 5010, New Zealand

*Email: [email protected]

Abstract A lateral flow assay (LFA) can provide a rapid and cost effective means to detect targets in-situ, however existing LFA formats (predominantly sandwich assays) are not suitable for small molecule targets. We present a new LFA design that probes the dissociation of aptamers from the surface of gold nanoparticles upon recognition of small targets. The target-induced removal of aptamer molecules from the surface of the coloured particles results in the particles being captured on a test line comprised of the protein bovine serum albumin immobilized on nitrocellulose. On the other hand, in the absense of target, aptamer coated particles are protected from capture on the test line and are instead captured at a control line comprised of the protein lysozyme. This protein is strongly positively charged under measurement conditions and therefore captures all gold nanoparticles, regardless of the presence of aptamers. The effectiveness and operation mechanism of this simply fabricated sensor was demonstrated by using a previously reported 35-mer aptamer for a small molecule, 17β-estradiol. The sensor exhibited nanomolar level of detection, excellent selectivity against potential interfering molecules, and robust operation in natural river water samples. The simplicity and performance of the sensor platform renders it applicable to a wide range of other aptamers targeting small molecules, as we demonstrated with a novel bisphenol-A aptamer. Additionally, we show that our LFA design is not confined to the specific proteins used as test and control lines, provided that their charge is appropriate to modulate the interaction with aptamer-coated or bare nanoparticles.

1. Introduction Lateral flow assay (LFA) methods have provided qualitative and semi-quantitative analysis for pathogens, drugs, hormones and metabolites in resource-poor or non-laboratory environments.1 These assays function when functionalised particles are transported by capillary wicking in porous substates

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and accumulate to form a visible line in the presence of an analyte. Gold nanoparticles (AuNPs) are frequently used for their intense colour, 2 and antibodies are widely used as the biorecognition probe owing to their ability to selectively bind to a target compound.3 Large analytes such as proteins and cells can be measured using a sandwich-type assay, whereby the target connects AuNPs to the test line via simultaneously binding to antibodies immobilised on both the test line and the AuNPs. 3,4 However, small targets - including steroids and toxins - are structurally less complex than proteins and are generally unable to form complexes with more than one antibody. Instead, small molecule targets are generally detected using antibodies in competitive assay formats.3,4 Moreover, in addition to their unsuitabliltiy for small molecule targets, antibodies themselves have a number of disadvantages as recognition elements, such as their high production cost and their instability in nonbuffered solutions and non-physiological environments.5 The development of simple LFA formats using alternative recognition elements, particularly for small molecule targets, is a topic of great commercial and academic interest. Aptamers, which are single stranded oligonucleotide sequences, have shown considerable success in analytical sensing, diagnostic, and drug delivery applications

6–8

since their discovery in 1990s.

9,10

Aptamers can be selected from synthetic combinatorial libraries of oligonucleotides through an in vitro selection process called SELEX to selectively bind a variety of targets including metal ions, small molecular weight targets,

12,13

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and large targets.14 Unlike antibodies, they undergo substantial

conformational changes upon target binding, enabling viable signal transduction via fluorescence, 15,16

size, 13 electrochemical, 8,17 and colorimetric 18–21 based mechanisms.

Aptamers have been successfully applied as an alternative to antibodies in sandwich type LFAs for a number of large molecular targets, where multiple binding interactions are possible, including thrombin,22 cancer cells,23 and bacterium.24 However, constructing aptamer based LFAs for small molecule targets – which can only bind to one aptamer at a time – is more challenging, requiring indirect signal transduction mechanisms to be engineered. In one previous example, a signal-on LFA was reported for adenosine and cocaine25 in which the target molecule induces disaggregation of crosslinked AuNPs from the conjugation pad, enabling the monomeric particles to flow and be captured via the biotin-streptavidin interaction at the test line. In order to crosslink the initial AuNP aggregates, adenosine or cocaine aptamers were extended with additional sequences such that an optimal amount of the aptamer sequence is engaged in cross-linking between two types of DNA-functionalized AuNPs, with enough overhang to initiate interaction with the target. The success of this assay requires preparation of various labelled particles and careful sequence design, requiring good knowledge of the aptamer-target interaction. These limitations may explain the high mM detection limit observed with this assay.


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Another example of an aptamer based LFA for small molecules is the competitive (inhibition) assay reported for ochratoxin A. Here, the aptamer was covalently tethered to AuNPs or fluorescent particles and target recognition competes with the hybridisation of the aptamer with a complementary DNA sequence immobilised on the strip.26,27 The slow DNA hybridization kinetics (at least 50s 28) hinders the applicability of this sensing design to a LFA where all steps must occur within several minutes. Additionally, competitive assays are generally not favoured due to their data interpretation difficulty, since the intensity of the test line is inversely proportional to the target concentration.1 A different competitive LFA was reported for the small molecule aflatoxin B1.29 Here, a biotin-modified aptamer was immobilised on streptavidin line, and recognition signals were visualised by the release of fluorophore (Cy5)-modified complementary DNA strand previously hybridised to the aptamer. The control line was made of an antibody for Cy5 dye. In addition to the drawbacks associated with interpreting competitive assays, this LFA also suffers from the need to read fluorescent signals using a strip reader, as well as the complexity of associated with using antibodies in addition to different DNA sequences. To overcome these limitations of aptamer-based LFAs for small molecules, herein we report a simple LFA format where non-specific adsorption to AuNPs is used to determine whether or not aptamers are bound to their target. This relies on the established observation that target binding can induce desorption of aptamers from the surface AuNPs because a large fraction of the bases is committed to a target-binding secondary structure rather than binding to the AuNP. This phenomenon that has been extensively exploited for solution-based colorimetric sensing, particularly for small molecule targets, where target recognition followed by salt addition triggers AuNP aggregation (and therefore color change). 19,21,30–33 Here, we have devised a way to identify when aptamers have desorbed from AuNPs via the surface interactions between AuNPs and proteins deposited directly as sharp lines on a nitrocellulose strip. Specifically, we use a test line comprised of a protein that is negatively charged in the desired conditions (e.g., biological pH) and with strong affinity to gold surfaces, such as bovine serum albumin (BSA). In the absence of target, the negatively charged aptamers coating the AuNP surface prevents the particles being captured on the test line. In contrast, when the target binding removes the aptamer coating from AuNPs, the particle surfaces are exposed and can be captured by the protein. The control line of the assay strip is constructed from a positively charged protein, lysozyme (Lys), which interacts indiscriminately with all AuNPs, whether or not they are still coated with aptamers, as indicated in Scheme 1. This LFA format retains the simplicity of the widely used colorimetric assays, while avoiding the need to identify the optimum salt concentration to trigger the colour change, which is a serious constraint for real samples.



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Scheme 1 Lateral flow sensor for small molecules with bovine serum albumin (BSA) as test line and Lysozyme (Lys) as control line, both pre-adsorbed on nitrocellulose (NC). The recognition of the target stimulates the dissociation of the aptamer sequences from the surface and results in subsequent formation of visible test line from the capture of uncovered AuNPs by BSA. The control line is always formed as a result of the strong interaction between AuNPs and Lys, regardless of whether the aptamer is still present. In this article, we use the proposed LFA format to detect 17β-estradiol (E2) using a 35-mer aptamer that we previously found to be effective in a AuNP based aggregation colorimetric sensor format.21 There is a pressing need for low cost and effective detection means for endocrine disrupting compounds like E2 accumulating in the environment,34,35 which is presently accomplished by complicated chromatographic and mass spectrometric analytical techniques.

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The sensor reported

herein responds to E2 with a 50 nM level of detection (in spiked distilled water and river water) and excellent selectivity against potential interfering molecules. We confirmed the generality of our scheme for other small molecule targets by applying it to an aptamer specifically targeting bisphenolA, and also confirmed that other suitable materials can be used as test and control lines, with appropriate consideration of their charge.

2. Experimental Section Reagents and Chemicals. 17β-estradiol (E2), progesterone, (P4), testosterone (T), bis(4hydroxyphenyl) methane (BPF), bisphenol A (BPA), chloroauric acid (HAuCl4), bovine serum albumin (≥ 98% lyophilized powder, MW = 66430 g.mol-1), Lysozyme (lyophilized powder MW=14 300 g.mol-1), and streptavidin were purchased from Sigma-Aldrich. E2 35-mer aptamer, BPA 75-mer aptamer, random 75-mer ssDNA, and random 35-mer ssDNA were purchased from Alpha DNA 4 ACS Paragon Plus Environment

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(sequences are provided in the supporting information). The ssDNA preparations were dissolved in deionized water (Milli-Q) and kept at −5 °C before use. Deionized water (Milli-Q, 18.2 MΩcm) was used in all experiments (unless stated), and all other chemicals were of analytical grade. Material Starter Kit that contains various nitrocellulose membranes, absorbent pads, and backing materials was purchased from DCN Diagnostics. Methods. AuNPs (10 nm in diameter, TEM image in Figure S1, Supporting Information) were synthesised according to a previously described protocol.

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The concentration of AuNPs was

estimated to be 14 nM using the Beer−Lambert law assuming an extinction coefficient of 2.7 × 108 M−1 cm−1 at 525 nm.38 AuNP−Aptamer recognition labels. Before exposure to aptamers, the amount of citrate stabilizer had to be reduced in order to make colloidal stability more sensitive to the presence of aptamers on their surfaces. This was achieved via 1:10 dilution of AuNPs in deionized water (Milli-Q), centrifugation at 12,500 rpm for 15 min (MIKRO 120-Hettich), and then resuspension to the initial concentration in deionized water (Milli-Q). Three mL of the purified AuNPs was immediately mixed with 0.3 nmoles of the desired ssDNA (aptamers or random sequences) to yield a concentration of 100 nM and a ssDNA/particle ratio of 9:1, for a particle number of 2.5 × 1013. AuNP-aptamer blend was immediately used for sensing after adding 10 mM NaCl. This particular salt concentration was chosen since it facilitates target sensing but does not induce AuNP aggregation that will adversely influence particles flow through the pores of NC strips (refer to Figure S9 in the supporting information). The successful ssDNA aptamer adsorption on the surface of AuNPs was confirmed via salt titration experiments, as shown in Figure S2 of the Supporting Information. In-solution characterisation of protein interaction with AuNPs. One mL of AuNPs at a concentration of 7 nM was used to study in-solution interaction of AuNPs and AuNP-aptamer with BSA and Lys. AuNPs and AuNP-aptamer were titrated with different concentrations of BSA and Lys by taking different volumes from their stock solutions, and maintaining the overall volume. The mixtures were left for 10 minutes and UV-Vis spectra were collected. BSA interaction was characterized based on red shift (nm) and Lys interaction was characterized based on aggregation (reported as the absorption ratio A625nm/A525nm). Lateral flow membranes. Nitrocellulose strips were cut into 5 cm × 0.5 cm sections using paper scissors. The absorbent pads were cut into 1 cm × 0.75 cm and laminated to the nitrocellulose membrane as shown in Scheme 1 and Figure 1c. The strips were washed with 100 µL 38.8 mM trisodium citrate to produce sharper and more defined test and control lines. After drying for 1 hour at room temperature, the test line was created at a distance of 2.5 cm from the strip’s lower end and separated with 0.5 cm from the control line. The test and control lines were made by carefully dropping 3 µL of the optimised BSA and lysozyme concentrations along the width of the strips. For test line, 0.1 g of BSA was dissolved in Mill-Q water to make a concentration of 1.5 µM. This solution was further diluted to 0.3 µM. 3 µL of this solution was deposited on the strips as test line. 5 ACS Paragon Plus Environment


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The number of deposited molecules on the strip is 2.34 × 1014 or 9.2 × 1012 mm-2 (assuming the liquid covers 5 × 5 mm2). Instead of BSA, streptavidin was used as test line for proof of generality of the developed sensor. 0.1 g of Streptavidin was dissolved in Mill-Q water to make a final concentration of 4.7 µM. Three µL of streptavidin solution (and from a different concentration of 1.6 µM) was deposited on nitrocellulose membranes to make test lines. For control line, 0.1 g of lysozyme was dissolved in Mill-Q water to give a solution of 7 µM concentration. Three µL of the desired Lys concentration was deposited on the strips as a test line. The number of deposited molecules on the strip is 5.4 × 15 (2.2 × 14 mm-2). Target Detection. Prior to deposition on the functionalized NC strips, E2; interfering agents; and BPA were incubated in solution with aptamer functionalized AuNPs (either E2-aptamer, BPAaptamer, or random sequences). These experiments were initially conducted using deionized water (Milli-Q). Stock solutions of the targets were made in ethanol before adding appropriate volumes to the water and adjusting the final ethanol content to 5%, ensuring sufficient target solubility. 20 µL of the resulting test samples was added to 100 µL of AuNP−aptamer solution to obtain different E2 concentrations in a total volume of 120 µL. Control samples comprised of blank water containing 5% ethanol. Samples were then incubated for a given amount of time (optimized as shown in Figure 2a) at room temperature to facilitate binding to the target. The same protocol was conducted with the experiments of sensing targets in river water. Water was collected from the Hutt River, Wellington, New Zealand, and pretreated via stirring 50 mL overnight at room temperature with 1 g of activated charcoal to eliminate any traces of organic compounds, including estrogenic compounds, and filtered twice with 0.22 µm syringe filters. Finally, 50 µL of AuNP-aptamer + target samples were deposited at the lower end of the strips and allowed to migrate through the NC membrane and across the test and control lines via capillary wicking action. Data analysis. Photos of assay strips were taken using a Nikon D3100 camera. The test line bands were analyzed with ImageJ program. The corresponding integrated band intensities were used for further analysis of the sensor performance.

3. Results and Discussion Our LFA design in Scheme 1 avoids the drawbacks of applying aptamer probes to sandwich and competitive assays, including the need to engineer and label aptamer sequences. Instead, we rely on the different non-specific binding affinities of aptamers (with and without the target) and different proteins to the surfaces of AuNPs. In order to validate the assumptions in the LFA design, we first investigated the interactions between the selected proteins and AuNPs – with and without aptamer coatings – in solution.


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3.1 Interaction of proteins with AuNPs in solution BSA was chosen to construct the test line due to its demonstrated use in model systems focused on the interaction with AuNPs,39 its commercial availability in bulk quantities, and its sufficient stability in conjugated systems.40–42 BSA can bind strongly to form stable conjugates with AuNPs;39,43–45 reported dissociation constants range from 1µM

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to 600 µM

39

and are highly sensitive to conditions and

surface modification. It was suggested that the electrostatic interaction between lysine amino acid on the BSA surface and citrate ions of AuNPs could be the governing interaction of the BSA adsorption.44,45 It should be noted that the thiol mediated adsorption facilitated by the unpaired-thiol group of BSA was previously ruled out.39 Indeed, the interaction of BSA and AuNPs can be completely eliminated by pre-adsorbing a surfactant like poly(ethylene glycol) on the surface of the AuNPs.46 Consistent with the aforementioned studies, we resolve strong interactions between bare AuNPs and BSA in solution using UV-vis spectroscopy of colloidal solutions. Figure 1a shows that adsorption of BSA induces a red-shift of the AuNP surface plasmon resonance peak (increase in full width half maximum of peak absorption values as shown in the figure inset) due to the formation of a dense dielectric layer that changes the local dielectric environment of the AuNP.

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Figure 1a shows the

normalised absorption spectra as a function of BSA concentration, with fixed AuNP concentration of 7 nM. The surface plasmon peak gradually redshifts upon titrating BSA from 1 nM to 10 nM, beyond which the redshift saturates, indicating that the surface is fully occupied with BSA.

As well as requiring exposed AuNP surfaces to bind to BSA, our sensor scheme also requires that aptamer-coated AuNPs do not bind to BSA. We also carried out a BSA titration against AuNPs that had been pre-adsorbed with a 35-mer E2 aptamer (3 aptamers/particle). Figure 1a shows that in contrast to the bare AuNPs, the absorption spectra of the aptamer-coated AuNPs do not further shift when BSA is added, indicating that the aptamer coating prevents BSA from adsorbing to AuNPs. It should be noted that adsorption of the aptamer results in a 5 nm redshift, again due to perturbation of the dielectric environment on the AuNP surface and Figure 1a shows the normalised data. This passivation can be understood as arising from aptamer molecules saturating the active sites responsible for BSA adsorption,39,43 as well as from the electrostatic repulsion between aptamers and BSA, both of which are negatively charged at neutral pH. Lysozyme was selected to form the control line due to its positive charge under neutral and biological pH conditions – making it likely to bind to AuNPs, regardless of whether or not aptamers coat the surface. The inset of Figure 1b shows that adding lysozyme to AuNPs or AuNP-aptamer conjugates in solution causes an obvious color change; The 525 nm surface plasmon band is replaced by a broader absorption band at 625 nm that is characteristic of AuNPs aggregation.

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Figure 1b shows

the 625 nm / 525 nm absorption ratio as a function of increasing Lys concentration. Although the AuNP-aptamer conjugate shows a greater degree of resistance against Lys-induced aggregation, both 7 ACS Paragon Plus Environment


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samples undergo a strong interaction with Lys that results in their aggregation. These results are consistent with previous work showing that Lys triggers AuNP aggregation, even when the particles are coated with large proteins. 47

Figure 1 A: Uv-vis characterisation of the interaction of BSA with AuNPs and AuNP-aptamer via the red shift of the AuNPs. B: Uv-vis characterisation of the interaction of Lys with AuNPs and AuNPaptamer via the absorption ratio A625/525 which is indicative of aggregates formation.

C:

performance of the developed assay strip with the optimised BSA and Lys concentrations (100 µM and 6900 µM respectively) with bare AuNPs (top strip) which result in appearance of two lines,


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AuNP-aptamer (middle strip) resulting in the formation of only control line, and AuNP- aptamertarget (bottom strip) which retain the test line and report the presence of the 500 nM of E2.

3.2 Strip based interaction of proteins with AuNPs and AuNP-aptamer Having established that the BSA-AuNP interaction is highly dependent on the presence of aptamers, whereas the Lys-AuNP interaction is indiscriminate, we proceeded to apply these proteins as test and control lines in a LFA. Note that this implementation of an LFA does not include a conjugate pad for direct exposure to the sample, but rather starts from a pre-mixed dispersion of AuNPs being applied to the strip. We screened a number of nitrocellulose membranes for their suitability, noting that the membrane must not alter the pH of the test samples, which may promote spurious interactions or induce AuNP aggregation. Additionally, membranes that are heavily coated with detergents or other additives might mask the desired non-specific adsorption interactions between AuNPs or AuNP-aptamer conjugates and the proteins forming test and control lines. Results from the nine candidate nitrocellulose membranes we screened are shown in Figure S3, each having different combinations of pore size and flow rate, surface coatings, and pH values. Only two membranes, designated as Prima 60 and Prima 125, exhibited the desired protein-particle interaction. Prima 60 gave consistent results and therefore was adopted for further experiments. Figure 1c demonstrates that our solution-based observations of AuNP-protein interactions are indeed translated to the assay strips. AuNPs (top strip) are mostly captured by the BSA and only those particles that survive the test line are captured by the Lys in the control line. However, AuNP-aptamer conjugates (middle strip) pass through BSA without being captured and migrate further to form a strong control line. Furthermore, pre-incubation of AuNP-aptamer with 500 nM of the target (E2) causes aptamer sequences to dissociate from the surface of AuNPs and subsequently form a visible coloration on the test line (bottom strip) confirming the feasibility of our selective gating strategy.

3.3 E2 lateral flow aptasensing Having established that BSA and Lys fulfil the main requirements of test and control lines, respectively, we proceeded to optimize their deposition. Excessively high concentrations of BSA can capture AuNP-aptamer conjugates, resulting in background signal in the test line that ultimately obscures target detection (Figure S4). In contrast, insufficient BSA will not capture AuNPs where a low concentration of the target has partially removed the aptamer coating. As can be seen in Figure S4, balancing these considerations led to an optimal concentration of the BSA deposition solution (100 µM). In the case of the Lys control line, no such thorough optimisation of the deposition solution



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was necessary. Figure S5 shows that a high concentration of Lys (6900 µM) ensures that all AuNPs are captured to form a sharp line.

Sensors that rely on aptamer recognition elements require the assay time to be optimized to allow for aptamers to interact with their target. In the present sensor, like with colorimetric sensors, further time may also be needed to ensure that target-bound aptamers have desorbed from the surface of AuNPs. To gain the optimum operation time for E2 detection, kinetic experiments were done by premixing E2 at a concentration of 1000 nM in dispersions of aptamer coated AuNPs for a given incubation time before running this dispersion along a pre-patterned strip. As can be seen in Figure 2a, the test line was not visible when no incubation time was allowed, and its intensity increased to its maximum at around 15 minutes incubation. Thus, 15 minutes incubation time was adopted for all sensing experiments.


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Figure 2 A) Optimizing the sensing time with 1000 nM of E2 premixed with AuNP-aptamer and assayed using the optimised concentration of BSA and Lys. 15 minutes assay time associated with the maximum signal and therefore was selected for further sensing experiments. B) Determination of level of detection, linearity, and reproducibility of E2 LFA by reporting integrated intensity extracted from ImageJ analysis of the test lines. Figure insets are representative images of the sensor response to various concentrations of E2. A control experiment was performed by exposing the same range of E2 concentration to a random 35-mer DNA previously adsorbed on AuNPs (circle data points and data and images labelled as random 35-mer). C) Specificity examination of E2 LFA against potential interfering molecules examined at a concentration of 1000 nM and compared to E2 signal. Error bars in B and C represent the STD of the mean of three independent experiments.



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Having established the optimal amount of BSA, Lys, and the incubation time for E2 sensing, we proceeded to perform the proposed LFA for E2. The photographs in Figure 2b reveal that the test line accumulates colour from the lowest E2 concentration used (50 nM), and continues to intensify with higher E2 concentrations. We used the ImageJ image analysis program to quantify test line intensities and to evaluate various sensing parameters including level of detection, linearity, and reproducibility. As can be seen in Figure 2b, E2 can be detected as low as 50 nM with a linear response up to 1000 nM, after which saturation occurs. A detection limit of 50 nM is calculated by extrapolating the linear response to a background of 3 × signal-to-noise level. This background was ascertained from a control assay where the 35-mer aptamer was replaced with a randomized 35-mer ssDNA sequence that does not interact with E2. The lack of signal from this randomized control confirms that the test line coloration in the sensor can be attributed to a specific aptamer-target binding interaction. Figure 2b also provides evidence that our LFA method for the detection of E2 is robust and reproducible, with error bars shown for three independent experiments starting from the adsorption of the aptamer on AuNPs.

We proceeded to examine the ability of our sensor to discriminate against structurally similar molecules potentially present in the test samples. These molecules were divided in two classes: (i) naturally existing hormones, progesterone (P4) and testosterone (T), which belong to the same steroidal family class as E2, and (ii) synthetic, nonsteroidal compounds such as bisphenol A (BPA) and 4, 4ʹ -methylenediphenol (BPF) (structures are shown in Figure S7), which can mimic the same estrogenic activity of the endogenous hormones. These molecules were examined under the same conditions as the E2 assay described above, with a concentration of 1000 nM, where the E2 sensor exhibits a strong response. Remarkably, the sensor shows excellent discrimination against the entire range of interfering molecules. It is worth noting that these results are consistent with our previous study with aggregation based colorimetric sensor using the same 35-mer aptamer when screening the same range of interfering molecules.21

The 50 nM level of detection places the assay sensitivity needed for the concentration range commonly encountered in environmental and water effluent samples.48,49 We examined the sensor in a more challenging fluid of spiked river water samples. Figure 3 shows that the sensor produces a similar response towards E2 spiked into river water samples as was found for double distilled water in Figure 2b. Similarly to E2 sensing in distilled water, we confirmed that the signals arose from a specific interaction by observing a lack of response when replacing the aptamer with the randomized 35-mer ssDNA. Again, we observed robust performance by repeating the sensing experiments of the same concentration range three times.


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Figure 3 A Performance of the developed LFA in spiked river water samples with a range of E2 concentration using the 35-mer aptamer and the 35-mer random ssDNA. B: ImageJ analysis of the test line of the strip presented in A. Error bars represent the STD of the mean of three independent experiments.

3.4 Generality of lateral flow aptasensor design The underpinning sensing mechanism should make our LFA method generally applicable to other small molecule binding aptamers, as has been shown for the mechanistically related colorimetric assay. 20,30,52 Moreover, other types of negatively charged proteins should also be able to fulfil the role of BSA. We confirmed this generality by constructing sensors whereby; a) the E2 aptamer was replaced with a 75-mer aptamer targeting bisphenol-A (BPA), or b) the BSA test line with a different negatively charged protein (streptavidin in this case). Prior to constructing the LFA with the 75-mer BPA aptamer, we ensured that this aptamer undergoes target-induced dissociation from AuNPs by performing the colorimetric assay in solution.21,53 Photographs and UV-vis spectra shown in Figure S6 confirmed BPA detection over the range of 50 1000 nM using the colorimetric assay, with only a baseline response for a control with a randomized sequence. Having confirmed that BPA aptamer can dissociate from the surface upon target binding, we optimized the LFA with this aptamer. Figures 4a and 4b show results of LFA with increasing concentration of BPA. The test line gained color from approximately 1000 nM BPA, and it continued to intensify with increasing BPA concentration. The level of detection of BPA using our LFA is of the 13 ACS Paragon Plus Environment


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same order of magnitude as the visual detection level of an aggregation based sensor using AuNPs previously reported with a different aptamer.

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It should be noted that the level of the colormetic

sensor based on AuNPs aggregation (50 nM, shown in Figure S6) is lower than the level of detection observed with LF assay (1000 nM shown in Figure 3). This could be attributed to the fact that additional optimization steps (e.g., reaction time, and BSA concentration) are needed when changing the LF assay recognition element. We again confirmed that our observations arose from specific target recognition by the lack of response when performing the assay with a random 75-mer ssDNA. Our results confirmed that our LFA design is not restricted to the 35-mer E2 aptamer. Additionally, the operation of the colorimetric sensor and LFA for the same aptamers illustrates that a strong relation exists between the two sensing methods; both have a common mechanistic origin.

Figure 4 A Performance of the developed LFA (BSA as a test line and Lys as control line) with the 75-mer BPA aptamer instead of E2 aptamer for proof of generality. B: ImageJ analysis of the test line of the strips presented in A.

We confirmed that other materials could fulfil the role that BSA plays as the test line by replacing BSA with a different negatively charged protein, streptavidin, which has a pI of ~ 5, and is therefore negatively charged at the working pH of 7.54 The concentration of streptavidin for deposition was optimized at 0.16 µM, as shown in Figure S8. Figure 5a demonstrates that streptavidin can 14 ACS Paragon Plus Environment

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successfully be used to probe the recognition of E2. Figure 5a and Figure 5b provide evidence that the same level of detection can be achieved for E2 when using streptavidin instead of BSA and the proposed sensor could be developed for a given target with a large range of negatively charged proteins once the relevant experimental conditions are optimised. Moreover, synthetic polymers, or even surface modifications of nitrocellulose, may also be used, provided that they have affinity towards AuNPs that can be modulated by the presence of negatively charged aptamers on the AuNPs.

Figure 5 Performance of LFA for E2 with streptavidin (0.16 µM) as a test line instead of BSA for proof of generality. A: strips representing the response of the LFA to increasing concentration of E2. B: ImageJ analysis (reported as relative integrated intensity) of the test line of the strips presented in A.

3.5 Comparison with previous literature The practicality and robust performance of our LF based sensor for E2 is apparent by comparing with the previous sensors for the same target. The 50 nM limit of detection of our LFA method (defined as S/N ˃ 3) is comparable to the visual detection limit of the previous aggregation based colorimetric sensor using the same 35-mer aptamer

21

and a different 76-mer split aptamer.50 Additionally, the

level of detection we report here is also comparable to various E2 sensors using different signal transductions such as size 13 fluorescence 16 and electrochemical. 51 As well as delivering comparable or better performance to these other small molecule sensors, our LFA sensor brings several practical advantages. Firstly, this method overcomes the significant variation encountered with the colorimetric sensor since the colorimetric detection mechanism relieses on balancing the system near a highly nonlinear and non-equilibrium aggregation threshold, which can result in false signals if the salt concentration is not optimally balanced. Secondly, the present LFA method does not require a UV-vis absorption spectrometer or other instrumentation to resolve low target concentrations. Although we 15 ACS Paragon Plus Environment


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used image processing to determine linearity and other parameters, the signals are easily resolvable by naked eye.

As shown in Table 1, in comparison to previously reported LFAs for small molecules, the 50 nM limit of detection of our sensor is at least three orders of magnitude lower than the LFA reported for cocaine and adenosine (0.02 mM),25 10 times lower than the lead LFA (500 nM),55 but higher than the LFAs reported for ochratoxin A (5 nM),26,27 and aflatoxin B1 (1 nM).29 Moreover, our dynamic range of nearly one order of magnitude is significantly wider than all reported LFAs, with most only operating within a narrow range of nM concentration. The operational target recognition time of our LFA is 18 min, which falls in the same time frame of other reported LFAs (Table 1). Successful implementation in real environmental or biological samples has been demonstrated for all the developed LFAs shown in Table 1. However, the generality of the developed LFA design was only demonstrated by the present study and by the cocaine and adenosine study.25 Furthermore, it is expected that implementing the present LFA design will significantly reduce the cost of manufacturing and performing the overall tests due to the following reasons: A) no aptamer modification (such as extension or thiol/fluorophores labelling) or additional complementary DNA sequences are required, and B) no additional antibody-antigen reaction is involved in the strip construction (such as biotin-streptavidin or Cy5 dye-antibody interaction), with only inexpensive and commercially available proteins such as BAS and Lys are required to construct the test and control lines. Finally, the present LFA avoids a number of limitations encountered by other LFAs, such as the additional purification steps required to prepare labelled particles, the requirement to identify the binding pocket responsible for target recognition, the need for a strip reader to generate signals, and the potential for false results from signal-off sensor designs.

Table 1 shows an apparent disadvantage the present implementation is that it requires a preincubation step before exposing the sample to the lateral flow strip. While a pre-incubation step could easily be accommodated in a packaged LFA test, it may also be possible to load the AuNP-aptamer conjugate to form a recognition pad on the strip by drying in an inert matrix (predominantly made of a protein like BSA, a surfactant like triton, a sugar like sucrose) like those used in most LFAs. Integrating the recognition step onto the LFA strip would require engineering the device to balance the flow kinetics with the aptamer-target binding kinetics.


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Table 1 Comparison between the LFA of the present work and other reported LFAs for the detection of small molecules. Target

LFA design

Detection

Dynamic

Detection

limit (nM)

range

time (min.)

Complexity and limitations

(nM) Adenosine

Disaggregation of cross-linked AuNPs

and cocaine

from

conjugation

pad,

4

2 × 10

monomeric

Not

Application

Generality

in real

demonstrated

Ref.

samples 5

Preparation

reported

and

purification of

labeled

25

particles. Requires aptamer modification as

particles flow and captured via biotin-

well as knowledge of binding pocket.

streptavidin interaction at test line. Lead

using

DNAzyme

Cleavage of cross-linked AuNPs from

500

conjugation pad, monomeric particles flow

and

captured

via

Not

18

Preparation

reported

particles.

biotin-

and

purification of

Requires

significant

labeled

-

55

-

26, 27

-

29

ssDNA

modification. Requires pre-incubation step.

streptavidin interaction at test line. Ochratoxin

Target

A

aptamer

recognition

competes

hybridisation

complementary

DNA

with

5

5- 150

10

Signal-off

with

response.

Requires

aptamer

hybridization to complementary sequence.

sequence

immobilised on the strip. Aflatoxin B1

Biotin-modified aptamer immobilised

1

1-100

30

Signal-off response. Requires fluorescent

on streptavidin line, recognition signals

strip reader. Involves the use of cy5

visualised by the release of fluorophore

antibody. Requires pre-incubation step.

-modified complementary DNA strand previously hybridised to the aptamer. E2 and BPA

Exploiting adsorption and desorption

50

50-1000

18

Requires pre-incubation step.

interactions of aptamers and proteins

This study

on AuNPs.

17

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Conclusions We have developed a new LFA platform for detecting small molecules using ssDNA aptamers. Starting from an established phenomenon where non-specifically adsorbed aptamers dissociate from the surface of AuNPs upon binding their target, we used the negatively charged BSA protein adsorbed on a nitrocellulose membrane to selectively probe whether aptamers were removed from the surface of AuNPs. Presence of the negatively charged aptamer prevents AuNPs from accumulating on the negatively charged BSA test line, whereas removal of the aptamers by interacting with the target allows the AuNPs to bind to the BSA and form a visible line. The control line was formed from a positively charged protein, Lys, which indiscriminately interacts with AuNPs regardless of whether aptamers remain. We demonstrated this LFA by detecting E2 with a comparable level of detection (50 nM) to previously reported aptasensors using various signal transductions, but without requiring any measurement instrumentation. Our sensor exhibited lower level of detection and wider dynamic range than most other published small molecule LFAs, along with comparable detection time. The sensor showed excellent discrimination against potential interfering molecules and robust operation in spiked river water samples. The simplicity of our LFA design compared with other small molecule LFAs makes the method generally applicable to other small molecule binding aptamers, which we demonstrated using a bisphenol-A aptamer, as well as to other negatively charged proteins, which we demonstrated using streptavidin. Our work provides a highly effective sensor scheme for the detection of small molecules, as well as a simple methodology that is broadly applicable to the growing demand for colorimetric aptasensors.

References (1)

Posthuma-Trumpie, G. A; Korf, J.; van Amerongen, A. Anal. Bioanal. Chem. 2009, 393 (2), 569–582.

(2)

Wilson, R. Chem. Soc. Rev. 2008, 37 (9), 2028–2045.

(3)

Ngom, B.; Guo, Y.; Wang, X.; Bi, D. Anal. Bioanal. Chem. 2010, 397 (3), 1113–1135.

(4)

Anfossi, L.; Baggiani, C.; Giovannoli, C.; D’Arco, G.; Giraudi, G. Anal. Bioanal. Chem. 2013, 405 (2–3), 467–480.

(5)

Chen, A.; Yang, S. Biosens. Bioelectron. 2015, 71, 230–242.

(6)

Farokhzad, O. C.; Jon, S.; Khademhosseini, A.; Tran, T.-N. T.; Lavan, D. a; Langer, R. Cancer Res. 2004, 64 (21), 7668–7672.

(7)

Meyer, C.; Hahn, U.; Rentmeister, A. J. Nucleic Acids 2011, 2011, 904750.

(8)

Zhu, B.; Alsager, O. a; Kumar, S.; Hodgkiss, J. M.; Travas-Sejdic, J. Biosens. Bioelectron. 2015, 70, 398–403.

(9)

Tuerk, C.; Gold, L. Science (80-. ). 1990, 249 (4968), 505–510. 18 ACS Paragon Plus Environment

Page 18 of 21

Page 19 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(10)

Ellington, A. D.; Szostak, J. W. Nature 1990, 346 (6287), 818–822.

(11)

Noeske, J.; Schwalbe, H.; Wöhnert, J. Nucleic Acids Res. 2007, 35 (15), 5262–5273.

(12)

Huizenga, D. E.; Szostak, J. W. Biochemistry 1995, 34 (2), 656–665.

(13)

Alsager, O. A; Kumar, S.; Willmott, G. R.; McNatty, K. P.; Hodgkiss, J. M. Biosens. Bioelectron. 2014, 57C, 262–268.

(14)

Tasset, D. M.; Kubik, M. F.; Steiner, W. J. Mol. Biol. 1997, 272 (5), 688–698.

(15)

Li, L.; Ge, P.; Selvin, P. R.; Lu, Y. Anal. Chem. 2012, 84 (18), 7852–7856.

(16)

Yildirim, N.; Long, F.; Gao, C.; He, M.; Shi, H.-C.; Gu, A. Z. Environ. Sci. Technol. 2012, 46 (6), 3288–3294.

(17)

Lin, Z.; Chen, L.; Zhang, G.; Liu, Q.; Qiu, B.; Cai, Z.; Chen, G. Analyst 2012, 137 (4), 819– 822.

(18)

Kim, Y. S.; Kim, J. H.; Kim, I. A.; Lee, S. J.; Jurng, J.; Gu, M. B. Biosens. Bioelectron. 2010, 26 (4), 1644–1649.

(19)

Zheng, Y.; Wang, Y.; Yang, X. Sensors Actuators B Chem. 2011, 156 (1), 95–99.

(20)

Yang, C.; Wang, Y.; Marty, J.-L.; Yang, X. Biosens. Bioelectron. 2011, 26 (5), 2724–2727.

(21)

Alsager, O. A.; Kumar, S.; Zhu, B.; Travas-Sejdic, J.; McNatty, K. P.; Hodgkiss, J. M. Anal. Chem. 2015, 87 (8), 4201–4209.

(22)

Xu, H.; Mao, X.; Zeng, Q.; Wang, S.; Kawde, A.-N.; Liu, G. Anal. Chem. 2009, 81 (2), 669– 675.

(23)

Liu, G.; Mao, X.; Phillips, J. A.; Xu, H.; Tan, W.; Zeng, L. Anal. Chem. 2009, 81 (24), 10013– 10018.

(24)

Bruno, J. Pathogens 2014, 3 (2), 341–355.

(25)

Liu, J.; Mazumdar, D.; Lu, Y. Angew. Chem. Int. Ed. Engl. 2006, 45 (47), 7955–7959.

(26)

Wang, L.; Ma, W.; Chen, W.; Liu, L.; Ma, W.; Zhu, Y.; Xu, L.; Kuang, H.; Xu, C. Biosens. Bioelectron. 2011, 26 (6), 3059–3062.

(27)

Wang, L.; Chen, W.; Ma, W.; Liu, L.; Ma, W.; Zhao, Y.; Zhu, Y.; Xu, L.; Kuang, H.; Xu, C. Chem. Commun. (Camb). 2011, 47 (5), 1574–1576.

(28)

Wei, C. W.; Cheng, J. Y.; Huang, C. T.; Yen, M. H.; Young, T. H. Nucleic Acids Res. 2005, 33 (8), 1–11.

(29)

Shim, W.-B.; Kim, M. J.; Mun, H.; Kim, M.-G. Biosens. Bioelectron. 2014, 62, 288–294.

(30)

Song, K.-M.; Cho, M.; Jo, H.; Min, K.; Jeon, S. H.; Kim, T.; Han, M. S.; Ku, J. K.; Ban, C. Anal. Biochem. 2011, 415 (2), 175–181.

(31)

Wei, H.; Li, B.; Li, J.; Wang, E.; Dong, S. Chem. Commun. (Camb). 2007, No. 36, 3735–3737.

(32)

Kwon, Y. S.; Ahmad Raston, N. H.; Gu, M. B. Chem. Commun. (Camb). 2014, 50 (1), 40–42.

(33)

Gopinath, S. C. B.; Lakshmipriya, T.; Awazu, K. Biosens. Bioelectron. 2014, 51, 115–123.

(34)

Diamanti-Kandarakis, E.; Bourguignon, J.-P.; Giudice, L. C.; Hauser, R.; Prins, G. S.; Soto, A. M.; Zoeller, R. T.; Gore, A. C. Endocr. Rev. 2009, 30 (4), 293–342.

(35)

SCHNEIDER, G.; KIRSCHNER, M. A.; BERKOWITZ, R.; ERTEL, N. H. J. Clin. Endocrinol. Metab. 1979, 48 (4), 633–638.

(36)

Auriol, M.; Filali-Meknassi, Y.; Tyagi, R. D.; Adams, C. D.; Surampalli, R. Y. Process 19 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochem. 2006, 41 (3), 525–539. (37)

Turkevich, J.; Stevenson, P. C.; Hiller, J. Discuss. Faraday Soc. 1951, 11 (c), 55–75.

(38)

Haiss, W.; Thanh, N. T. K.; Aveyard, J.; Fernig, D. G. Anal. Chem. 2007, 79 (11), 4215–4221.

(39) Dominguez-Medina, S.; Blankenburg, J.; Olson, J.; Landes, C. F.; Link, S. ACS Sustain. Chem. Eng. 2013, 1 (7), 833–842. (40)

Rezwan, K.; Meier, L. P.; Gauckler, L. J. Biomaterials 2005, 26 (21), 4351–4357.

(41)

Burton, W. G.; Nugent, K. D.; Slattery, T. K.; Summers, B. R.; Snyder, L. R. J. Chromatogr. A 1988, 443, 363–379.

(42)

Chen, Y.-M.; Yu, C.-J.; Cheng, T.-L.; Tseng, W.-L. Langmuir 2008, 24 (7), 3654–3660.

(43)

Tsai, D.; Delrio, F. W.; Keene, A. M.; Tyner, K. M.; Maccuspie, R. I.; Cho, T. J.; Zachariah, M. R.; Hackley, V. A. 2011, 2464–2477.

(44)

Casals, E.; Pfaller, T.; Duschl, A.; Oostingh, G. J.; Puntes, V. ACS Nano 2010, 4 (7), 3623– 3632.

(45)

Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Langmuir 2005, 21 (20), 9303–9307.

(46) Dominguez-Medina, S.; McDonough, S.; Swanglap, P.; Landes, C. F.; Link, S. Langmuir 2012, 28 (24), 9131–9139. (47)

Yi-Ming Chen, †; Cheng-Ju Yu, †; Tian-Lu Cheng ‡, §; and Wei-Lung Tseng* †, §. Langmuir 2008, 24 (7), 3654–3660.

(48)

Lee, H. B.; Peart, T. E. J. AOAC Int. 1998, 81 (6), 1209—1216.

(49)

Majima, K.; Fukui, T.; Yuan, J.; Wang, G.; Matsumoto, K. Anal. Sci. 2002, 18 (8), 869–874.

(50)

Liu, J.; Bai, W.; Niu, S.; Zhu, C.; Yang, S.; Chen, A. Sci. Rep. 2014, 4, 7571.

(51)

Olowu, R. a; Arotiba, O.; Mailu, S. N.; Waryo, T. T.; Baker, P.; Iwuoha, E. Sensors (Basel). 2010, 10 (11), 9872–9890.

(52)

Kim, Y. S.; Kim, J. H.; Kim, I. A.; Lee, S. J.; Gu, M. B. Biosens. Bioelectron. 2011, 26 (10), 4058–4063.

(53)

Mei, Z.; Chu, H.; Chen, W.; Xue, F.; Liu, J.; Xu, H.; Zhang, R.; Zheng, L. Biosens. Bioelectron. 2013, 39 (1), 26–30.

(54)

Robison, A. D.; Huang, D.; Jung, H.; Cremer, P. S. Biointerphases 2013, 8 (1), 1.

(55)

Mazumdar, D.; Liu, J.; Lu, G.; Zhou, J.; Lu, Y. Chemical Communications. 2010, 46(9), 14161418.

Supporting Information The following supporting information is available online: TEM images of AuNPs, sequences used in this study, salt titration experiments, selection of NC membrane, optimizing BSA; streptavidin; and Lys concentrations, BPA colorimetric sensing using AuNP-aggregation scheme, molecular structures of detected targets, and optimizing salt concentration for target sensing.


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Graphical abstract (3 inX 2 in)


Lateral Flow Aptasensor for Small Molecule ... - ACS Publications

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