Integration of Solution-Based Assays onto Lateral Flow Device for One

Aug 8, 2016 - The LFD system demonstrated here simplifies the POC operation significantly and thus allows an even wider application of the PGM for POC...
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Integration of solution-based assays onto lateral flow device for onestep quantitative point-of-care diagnostics using personal glucose meter Jing-Jing Zhang, Zhe Shen, Yu Xiang, and Yi Lu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00270 • Publication Date (Web): 08 Aug 2016 Downloaded from http://pubs.acs.org on August 8, 2016

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Integration of solution-based assays onto lateral flow device for one-step quantitative point-of-care diagnostics using personal glucose meter JingJing Zhang,† Zhe Shen,† Yu Xiang, †,‡ and Yi Lu*† † ‡

Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana IL 61801, USA Department of Chemistry, Tsinghua University, Beijing 100084 (P.R. China).

ABSTRACT: Personal glucose meter (PGM) has been recently adapted as a novel point-of-care (POC) device for the detection various targets. However, multi-step target binding and enzymatic reactions in solution make it difficult for end users to use this technology for POC detection. To overcome this limitation, we report herein the integration of all assay procedures in solution onto a lateral flow device (LFD) by demonstrating competitive assays in a single step for quantitative detection of both a small molecule (cocaine) and a large protein (streptavidin) based on the glucose measured by PGMs. The LFD system demonstrated here simplifies the POC operation significantly and thus allows an even wider application of the PGM for POC diagnostics at home or in lowresource settings.

KEYWORDS: Lateral flow, glucose meter, point-of-care, biosensors, aptamers

Point-of-care (POC) diagnosis can facilitate the detection of diseases or monitoring biomarkers related to human health at home or in the field.1-6 To this end, various POC devices have been developed in recent years, among which the personal glucose meter (PGM) is the most widely used device in the world because of its portable size, easy operation, low cost and reliable quantitative results.7 However, the traditional PGMs are designed to monitor only blood glucose levels.8 To overcome this challenge, Whitesides and co-workers have added more functionality to the basic PGMs by modifying the PGM strips by replacing the enzymes for glucose (e.g. glucose oxidase) with those enzymes selective for other target analytes (e.g. cholesterol, lactate, and alcohol).9 This approach, however, is still limited, as enzymes selective for every potential target may not be available and the modified strips can only be operated on a specific brand of PGMs. To broaden the applicability of PGMs, we and others have developed approaches to link the selective binding of non-glucose targets with aptamers, DNAzymes or antibodies through conjugation of these DNAzyme/aptamer/antibodies with an enzyme such as invertase or amylase, which can catalyze the conversion of non-glucose analytes into glucose.10-23 While the results are encouraging, the majority of these systems involve multiple sequential steps, including affinity capture on a solid surface (e.g. magnetic materials), physical separation, and chemical or biochemical signal amplifications, making it less user-friendly for POC applications. Some progress has been made towards the use of PGMs for enzyme activity assay in a single step; however, it either requires sophisticated syntheses of the enzyme substrates that are covalently linked to glucose or is not transferable as a general strategy.24-26 Recently, Wang and coworkers integrated a microfluidic chip with PGM to develop a quantitative approach for detection of DNA using PGMs.27 Despite the progress, it is desirable to develop a much simpler

and less expensive method for detecting not only DNA, but also a wide range of targets in a single step by users for selfdiagnostics at home. We herein report the integration of all the steps into a lateral flow device (LFD), similar to glucose strips, for quantitative POC analysis using PGM. The proposed PGM-LFDs system combines the advantage of both PGMs and LFDs, offering a new way for point-of-care diagnostics in a low-resource settings. LFDs have been widely used for medical diagnostics, ranging from home testing for pregnancy and drugs of abuse, to disease detection in low resource settings.28-35 It allows a single step operation with high speed, low operational cost, simple instrumentation in a user friendly format. The incorporation of chromatographic separation makes it possible to remove interferences and achieve high sensitivity.36-38 Despite these advantages, most of the LFD strips can achieve only qualitative or semi-quantitative detection.39-41 To make LFD more quantitative, the LDF strips have been interfaced with portable devices, such as colorimeters.42-46 However, the colorimetric detection method is often vulnerable to interference from biofluids, including the color of blood or urine. Electrochemical detection used in PGM, on the other hand, is much less vulnerable to such intrusions and is easier for electronic amplification.

Experimental Section Materials and reagents. Streptavidin-coated magnetic beads (1 µm in diameter) and Amicon-10K/100K centrifugal filters were purchased from Bangs Laboratories Inc. (Fishers, IN) and Millipore Inc. (Billerica, MA), respectively. Streptavidin, EZ-LinkTM NHS-PEG4-Biotin, and sulfosuccinimidyl-4(N-maleimidomethyl)-cyclohexane-1-carboxylate (Sulfo-

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SMCC) were obtained from Pierce Inc. (Rockford, IL). Grade VII invertase from baker’s yeast (S. cerevisiae), tris(2carboxyethyl)phosphine (TCEP), sucrose, and other chemicals for buffers and solvents were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). The commercial personal glucose meter used in this study was an ACCU-CHEK Avia. glucose meter, and the test of a solution by the meter was simply conducted by contacting the solution with the strip loaded on the glucose meter. Buffers used in this work: PBS Buffer: pH 7.3, 0.1 M sodium phosphate, 0.1 M NaCl, 0.05% Tween-20. DNA sequences used in the method were purchased from Integrated DNA Technologies, Inc. (Coralville, IA): from left to right: 5′to 3′ Biotin-modified DNA (Biotin-DNA) for cocaine, and uranium sensors: TCACAGATGAGTAAAAAAAAAAAA-biotin. Thiol-modified DNA (Thiol-DNA) for cocaine and uranium sensors: HS-AAAAAAAAAAAAGTCTCCCGAGAT. Cocaine aptamer (Coc-Apt): TTTTTTACTCATCTGTGAATCTCGGGAGACAAGGATA AATCCTTCAATGAAGTGGGTCTCCC

DNA-Invertase conjugation. The DNA−Invertase conjugate was synthesized by the maleimide-thiol reaction using heterobifunctional linker sulfo-SMCC (Figure S1). Briefly, 30 µL of 1 mM thiol-DNA, 2 µL of 1 M PBS buffer (pH 5.5), and 2 µL of 30 mM TCEP were mixed and incubated at room temperature for 1 hour. Then, the thiol-DNA was purified by Amicon-10K using PBS buffer 8 times. For Invertase conjugation, 400 µL of 20 mg mL-1 invertase in PBS buffer was mixed with 1 mg of sulfo-SMCC. After vortexing for 5 minutes, the solution was placed on a shaker for 1 hour at room temperature. The mixture was then purified by Amicon-10K using PBS buffer 8 times. The purified solution of sulfo-SMCCactivated Invertase was mixed with the above solution of thiolDNA. The resulting solution was kept at room temperature for 48 hours. To remove un-reacted thiol-DNA, the solution was purified by Amicon-30K 8 times using PBS buffer. The DNA−Invertase conjugate was finally dispersed in PBS buffer with a concentration of 20 mg mL-1. Biotin-invertase conjugation. The biotin-invertase conjugate was synthesized by using the NHS-ester biotinylation reagent (Figure S3). Briefly, to 1 mL 20 mg mL-1 invertase in PBS buffer without Tween-20, 5 mg EZ-Link NHS-PEG4Biotin was added and the mixture was well mixed at room temperature for 4 h. Then, the biotin-invertase conjugate was purified 8 times by Amicon-100K using PBS buffer without Tween-20, and then dispersed in 1 mL of PBS Buffer with a final concentration of 2.0 mg mL-1 for preparation of lateral flow strips. Preparation of DNA-Invertase conjugate functionalized MBs. For the cocaine sensor, a portion of 1

mL 1 mg mL-1 solution of MBs was placed close to a magnetic rack for 1 minute. The clear solution was discarded and replaced by 1 mL of PBS buffer. This buffer exchange procedure was repeated twice. Then, 12 µL 0.5 mM Biotin-DNA was added to the MBs solution and mixed well for 30 minutes at room temperature. After that, the MBs were washed twice using PBS buffer to remove excess biotin-DNA. Later, 12 µL of 0.5 mM cocaine aptamer were added to the MB solution and well mixed for 30 minutes at room temperature. After three times washing using PBS buffer to remove excess DNA, 400 µL of 20 mg mL-1 DNA−Invertase conjugate was added to

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the solution and well mixed at room temperature for 30 minutes. Excess DNA−Invertase conjugate was washed off by PBS buffer three times. The DNA−Invertase functionalized MBs were then dispersed in 1 mL of PBS Buffer for preparation of lateral flow strips.

Design and preparation of lateral flow strips. For all experiments, the Millipore High-FlowTM Plus Assembly Kit (Millipore Corporation, Bedford, MA) was used. The kit contains a High-Flow Plus Cellulose Ester Membrane with a nominal capillary flow time of 90 seconds/4 cm and a nominal membrane thickness of 135 µm directly cast onto 2 mil polyester backing and placed on an adhesive card. Each strip was cut from a pad and assembled according to the design using an office paper cutter. The detailed design of strip was shown in Figure 1A. The length of the filter membrane along the flow direction is 12.5 mm on the backing. To prepare the reaction pads, spot cellulose pads with 1 M sucrose water solution to ensure complete immersion and leave at room temperature to dry overnight. After drying, cut the reaction pads into proper dimensions as mentioned in Figure S4 (12 mm length, 4 mm width). To prepare the sample pad for cocaine detection, spot cellulose pads (34 mm length, 4 mm width) previously assembled on the backing pad with 10 µL DNA-Invertase conjugate functionalized MBs in PBS buffer and leave at room temperature for 15 minutes to dry (Figure S3). To prepare the sample pad for streptavidin detection, spot cellulose pads (34 mm length, 4 mm width) previously assembled on the backing pad with 10 µL of 2.0 mg mL-1 Biotin-Invertase conjugate and 10 µL of 10 mg mL-1 streptavidin-coated MBs (molecular weight ratio, streptavidin/biotin > 2:1), and then leave at room temperature for 15 minutes to dry (Figure S4). The sample pad, and reaction pad were attached to the adhesive card of the filter membrane in a way as shown in Figure S4. The overlap for each pad was ~3 mm, and the width was ~4 mm cut by a paper cutter. Target detection using lateral flow strips. For cocaine detection using LFDs integrated glucose meters, the LFDs coated with DNA-Invertase conjugate functionalized MBs were dipped into 200 µL of sample solution containing different concentration of cocaine in 1.7-mL Eppendorf tubes (Figure 1B). The capillary effect of the sample pad would drive sample solution up along the filter membrane into the sucrose pad. After 30 minutes of reaction, the solution in the sucrose pad was squeezed out and applied to a portable glucometer to quantify the concentration of glucose. For streptavidin detection using LFDs integrated glucose meters, the LFDs coated with Biotin-Invertase conjugate and streptavidin-coated MBs were dipped into 200 µL of sample solution containing different concentration of streptavidin in 1.7-mL Eppendorf tubes (Figure 1B). The capillary effect of the sample pad would drive sample solution up along the filter membrane into the sucrose pad. After 20 minutes of reaction, the solution in the sucrose pad was squeezed out and applied to a portable glucometer to quantify the concentration of glucose.

Results and Discussion Design and principle of LFD-based PGM sensors. The LFD was prepared following the similar protocol reported previously by our lab, with slight modification.47 Briefly, it

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consists of three overlapping pads placed on a backing (from left to right): sample pad (34 mm), HiFlow Plus filter membrane (12.5 mm), and reaction pad (12 mm) (Figure 1A). The invertase-functionalized magnetic beads (MBs) were spotted on the specific position of the sample pad, and sucrose as the enzyme substrate was applied on the reaction pad (Figure 1A). The whole device was then dried at room temperature before use. When the sample pad of the device was dipped into a solution (Figure 1B), the solution would move up along the device and the target inside could transfer the invertase from the sample pad to reaction pad where the sucrose would be hydrolysed to glucose. Although the optimal temperature for sucrose to glucose conversion catalysed by invertase from bakers’ yeast is 55 ºC,19 we performed all the assays at room temperature to simplify our LFD-PGM sensor for practical application. Since the produced glucose had a direct relationship with the amount of invertase released, which depends on the concentration of target in sample solution, the concentration of target could be quantified by monitoring the glucose using a PGM.

2C, the PGM signal increased with the increasing concentration of cocaine in the samples, and a limit of detection (LOD) of 7.7 µM, well within the detection range of 0–300 µM cocaine was achieved. This sensitivity is comparable to the commercial cocaine test kits, and previous reported LFDsbased cocaine sensors, 47, 50 but slightly worse than our previously reported in-tube based PGM assay for cocaine (LOD = 3.4 µM).10 The decreased sensitivity might be due to the loss of released DNA-Invertase conjugate before travelling into the reaction pad. To evaluate the selectivity of the LFD-PGM system for cocaine detection, the LFD was dipped into a solution containing either ADP, ATP, or Uridine. The PGM reading is much less than that in the presence of cocaine and is similar to that in just PBS buffer (as blank; Figure 2D). These results indicate that our LFD-PGM-system retained the aptamer selectivity with a highly specific response to the target, cocaine. As the cost of assembling such strips is very low (calculated to be at < $3 per test), the required time of reaction is short ( 2:1. The solution containing STV-biotin-INV would further travel into the reaction pad, where the INV catalyzed the hydrolysis of sucrose into glucose in amounts that was proportional to the concentration of STV in the solution. A PGM was then used to quantify the concentrations of glucose in the reaction pad. As shown in Figure 3B, the presence of STV in the samples resulted in elevated glucose production, and the concentration of STV in the sample was dependent on the glucose concentration measured by PGMs, showing an approximate linear relationship within 0−1.25 µM until the signal gradually reached saturation at 2.5 µM. The detection limit was determined to be 0.2 µM based on a 3σb/slope, where σb is the standard deviation of three blank samples. To investigate the potential LOD achievable in principle, we performed an assay that directly measured the lowest level of invertase-biotin conjugate on the reaction pad of the LFDs. As shown in Figure S5, the LOD of invertase-biotin conjugate was calculated to be 50 nM. Therefore, the potential LOD for streptavidin in principle was calculated to be 12.5 nM, assuming the invertase-release was 100% and each streptavidin could bind four invertase-biotin. This potential LOD is comparable to our previous in-tube based PGM assay for streptavidin (LOD of 4.0 nM), but much lower than the streptavidin assay on the proposed LFD-PGM format (LOD of 200 nM). This reduced sensitivity was due to the lower binding efficiency of streptavidin and biotin-invertase conjugate, as well as potential loss of the released biotininvertase on the strips before moving into reaction pad for sucrose to glucose conversion. In comparison with other detection methods, the distinct advantages of this proposed LFDPGM system were low cost (~ $2.5 per test), short reaction time (~ 20 min), and small sample volume (200 µL), which provides a facile, cost-effective and on-site method of quantification of STV under low-resource settings.13, 51

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Figure 3. (A) Proof-of-concept competitive assay for the detection of streptavidin by streptavidin-coated MBs and invertase-biotin conjugates using a PGM integrated with a lateral flow dipstick.(B) Relationship between PGM signals and streptavidin concentrations in the samples.

Conclusions We have developed a simple and general quantitative POC diagnostic system by integrating target recognition by DNA aptamer or biotin-STV with LFD for sample processing, and PGM for signal readout. Such LFD-PGM system allows the inexpensive, rapid, portable, and quantitative detection of a wide range of targets in a single step, thereby offering several advantages. First, compared with previous PGM-based sensors, the employment of LFDs helps to improve and simplify the sample testing, decrease the total sample-to-answer assay time, and make it suitable for a rapid diagnostics. Second, the wide availability, operation simplicity, high accuracy, and low cost of the pocket-sized PGMs to end users provide a simple but reliable tool for quantitative signal capture and collection. Moreover, as a variety of functional DNAs or antibodies for a broad range of targets are either available or can be obtained through SELEX,52-53 the method developed here can be used as a powerful tool for the detection of other targets, simply by changing specific molecular recognition elements on the LFD strips. These advantages, combined with FDA-approved PGM can be interfaced with smartphones for wireless transmission of the data. We expect that the LFD-PGM system demonstrated here will become a promising alternative POCT system designed for low-resource settings.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.xx.. Supporting information for se-2016-002705.docx: This Supporting Information provides additional data on the mechanism of DNA and invertase conjugation (Figure S1), the fluorescence assay of cocaine (Figure S2), the mechanism of biotin and invertase conjugation (Figure S3), the scheme of assembly of a lateral flow device (Figure S4), and the glucose meter response to invertase-biotin conjugate (Figure S5).

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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ACKNOWLEDGMENT We wish to thank Ms. Nitya Sai Reddy Satyavolu for help with proof-reading and the U.S. National Institutes of Health (ES16865) for financial support.

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