Integrating Target-Responsive Hydrogel with Pressuremeter Readout

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Integrating Target-Responsive Hydrogel with Pressuremeter Readout Enables Simple, Sensitive, User-friendly, Quantitative Point-of-Care Testing Dan Liu, Shasha Jia, Huimin Zhang, Yanli Ma, Zhichao Guan, Jiuxing Li, Zhi Zhu, Tianhai Ji, and Chaoyong James Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b05531 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 28, 2017

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ACS Applied Materials & Interfaces

Integrating Target-Responsive Hydrogel with Pressuremeter Readout Enables Simple, Sensitive, User-friendly, Quantitative Point-of-Care Testing Dan Liu, Shasha Jia, Huimin Zhang, Yanli Ma, Zhichao Guan, Jiuxing Li, Zhi Zhu*, Tianhai Ji, Chaoyong James Yang*

State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Center of Chemistry for Energy Materials, Key Laboratory for Chemical Biology of Fujian Province, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Affiliated Chenggong Hospital, Xiamen University, Xiamen 361005, P. R. China. * To whom correspondence should be addressed. Tel: (+86) 592-218-7601; Fax: (+86) 592-2189959. E-mail: [email protected]; [email protected]

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ABSTRACT:

Point-of-care testing (POCT) with the advantages of speed, simplicity, and low cost, as well as no need for instrumentation is critical for the measurement of analytes in a variety of environments lacking access to laboratory infrastructure. In the present study, a hydrogel pressure-based assay for quantitative POCT was developed by integrating a target-responsive hydrogel with pressuremeter readout. The target-responsive hydrogels were constructed with DNA grafted linear polyacrylamide and the crosslinking DNA for selective target recognition. Hydrogel response to target substance allows release of the preloaded Pt nanoparticles, which have good stability and excellent catalytic ability for decomposing H2O2 to O2. Then, the generated O2 in a sealed environment leads to significant pressure increase, which can be easily read out by a handheld pressuremeter. Using this target-responsive hydrogel pressure-based assay, portable and highly sensitive detection of cocaine, ochratoxin A and lead ion were achieved with excellent accuracy and selectivity. With the advantages of portability, high sensitivity, and simple sample processing, the hydrogel pressure-based assay shows great potential for quantitative POCT of a broad range of targets in resource-limited settings. KEYWORDS: target-responsive hydrogel, pressuremeter readout, quantitative point-of-care testing, Pt nanoparticles, gas generation


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Introduction The development and popularization of Point-of-Care Testing (POCT) is of vital importance in healthcare diagnostics, food safety assessment and environmental monitoring, especially in resource-limited settings.1-5 To meet the need for POCT, developers need to consider the simplification of signal transduction strategy that converts the target recognition event into measurable parameters.6-8 Currently, the measurement for POCT generally utilizes immunoassay systems, such as immune colloidal gold technique (ICG), lateral flow assay (LFA) and enzymelinked immunosorbent assay (ELISA).9-11 The above tests can be carried out by converting protein recognition into absorbance or fluorescence signals. However, immunoassay strategies have several limitations. For instance, antibody-based detection is generally restricted to protein targets, and the activity of the antibody can be easily influenced during the modification process. Therefore, new signal recognition elements that allow simple sample preparation with the capacity of detecting a wide variety of analytes are urgently needed to develop extensive POCT platforms. With the advantages of high stability, broad range of target molecules, and easy synthesis, functional nucleic acids act as recognition elements have received extensive attraction in the development of biosensing devices in recent decades.12,13 Functional nucleic acids generally include aptamers14 (which specifically bind targets) and DNAzymes15 (also known as DNA enzymes). Aptamers/DNAzymes can be artificially generated against a broad range of targets, from small metal ions and organic molecules to biomolecules, and even whole cells, by in vitro selection method.16 Many aptamer/ DNAzyme based POCT sensors have been developed for various targets, such as small molecules,17 ions,18 proteins,19 and pathogens,20 with qualitative or semi-quantitative detection that requires no instrumentation. Lu’s group recently reported an


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portable and quantitative method that combines functional DNA probes with a glucose meter for a broad range of targets analysis, specially enabling cocaine to be detected with a detection limit of several µM.21,22 DNA-cross-linked hydrogels with synthetic polymers as backbone and DNA as cross-linker are regarded as an ideal signal transduction strategy for POCT due to their simplicity, stability, portability, and ease of storage.23 Specifically, portable detection can be accomplished by a volume or phase transition of target-responsive smart DNA hydrogels in response to target and DNA recognition. The catalysts trapped inside the hydrogel possess high catalytic efficiency and great stability. Because the smart DNA hydrogel design is based on DNA hybridization, the hydrogel strategy can be further extended to a variety of targets that have specific aptamer sequences. DNA hydrogels have been broadly used for biosensing,24-27 target capture and release,28 and even cell adhesion.29,30 Specially, we have integrated DNA hydrogels with glucometer readout, paper chips and volumetric bar-chart chips (V-chip) for quantitative POCT.31-38 While the glucometer and paper chips have relatively low sensitivity, the V-Chip also suffers from several limitations. First, the V-chip is made of two pieces of frangible glass, and requires photolithography during fabrication with high cost and a tedious assembly process before utilization. Second, many pre-processing and washing steps are needed to reduce nonspecific adsorption. Third, during the repeated usage of the V-Chip, well-trained staff are needed to deal with piranha solution (extremely corrosive, reactive, and potentially explosive), setting a particularly significant barrier for general population. A highly senstive, inexpensive, user-friendly signal readout device is needed for integration with DNA hydrogels to facilitate their application in resource-limited settings.


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Very recently, our research group has developed pressure-based bioassays (PASS) for quantitative POCT using a portable pressuremeter readout.39,40 The PASS method translates molecular recognition events (target binding by an antibody) into an easily measurable pressure parameter. In the present study, we report combination of PASS with a smart hydrogel encapsulating Pt nanoparticles (PtNPs) for simple, senstive and user-friendly POCT of a variety of toxic substances. Hydrogel response to target substances allows release of PtNPs, which have high catalytic activity and stability superior to natural enzymes.39 The released PtNPs can effectively catalyze the decomposition of H2O2 to generate abundant O2, leading to a significant pressure increase, which can be easily read out by the pressuremeter. Using this strategy, cocaine, ochratoxin A (OTA) and heavy metal lead ions were detected with excellent accuracy and selectivity.

Materials and methods Materials and Reagents. Cocaine was acquired from the National Institutes for Food and Drug Control (Beijing, China). Ochratoxin A was purchased from Pribolab (Singapore). Ammonium persulfate (APS), N,N,N',N'-tetramethylethylenediamine (TEMED) and acrylamide were purchased

from

Sigma-Aldrich

(St.

Louis,

MO,

USA).

2-Cyanoethyl

diisopropyl-

chlorophosphoramidite was purchased from Chem Genes (Wilmington, MA, USA). DNA synthesis reagents were purchased from Glen Research (Sterling, VA, Germany). Other reagents were purchased from Sinopharm Chemical Reagent (Shanghai, China). Buffer containing 23 mM NaH2PO4, 77 mM Na2HPO4, 50 mM NaCl, 5 mM MgCl2 (pH 7.3) was used for cocaine detection. A buffer consisting of 10 mM Tris-AcOH buffer, 300 mM NaCl (pH 8.0) was used for


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Pb2+ detection. A buffer with 10 mM Tris-HCl, 25 mM KCl, 120 mM NaCl, and 20 mM CaCl2 (pH 8.5) was used for OTA detection. Preparation of PtNPs. The PtNPs were synthesized as reported previously.41 Briefly, an aqueous solution of ascorbic acid (100 µL, 0.4 M) was added to 1 mL of 1 mM H2PtCl6 and immediately incubated at 80°C for 30 min. The synthesized PtNPs were stored at 4°C before use. PtNP is a critical material for the detection system works as O2 producing catalyst. The decomposition of H2O2 is an exothermal reaction, and many catalysts have been founded for this reaction, such as metal ions, enzyme and metals. Among them, platinum has been studied for a long time and strongly catalyzes the decomposition of H2O2 into H2O and O2. Typically, this is a two stages reaction. First, the binding of H2O2 to platinum surface produce OH radical. Secondly, OH radical is oxidized to O2 by the platinum oxide on platinum surface.42,43 Then O2 will oxidize the platinum surface again, which promote more O2 production. Synthesis of Acrylic-DMT Phosphoramidite. In order to graft DNA strands onto the linear polyacrylamide, acrylic-DMT phosphoramidite was synthesized as shown in Scheme S1. The entire synthesis process was reported in previous work.44 First, acrylic-(OH)2 was synthesized via reacting methacrylic acid (176 mg, 2 mmol) and 6-amino-2-hydroxymethylhexan-1-ol (294 mg, 2 mmol) overnight at room temperature. Then, the acrylic-(OH)2 (270 mg, 1.17 mmol) was reacted with DMT-Cl (476 mg, 1.40 mmol) to produce acrylic-DMT. Afterwards, acrylic-DMT (320.7

mg,

0.62

mmol)

in

anhydrous

CH2Cl2

(1

mL)

was

reacted

with

2-

cyanoethyldiisopropylchloro-phosphoramidite (0.23 mL, 0.74 mmol) at 0°C. Each compound was purified by silica gel column chromatography. DNA Synthesis and Purification. According to the DNA synthesis protocol, all oligonucleotides (Table S1) were synthesized using a PolyGen 12-Column DNA Synthesizer.


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Strands A and B were then modified with the acrylic-DMT phosphoramidite at the 5’-end. The products were cleaved from the solid support, deprotected by the mixture of methylamine ammonia (v/v, 1:1) treatment, and purified using an Agilent (Santa Clara, CA, USA) 1100 series HPLC system with a reversed-phase C18 column. Triethylammonium acetate solution (0.1 M, pH 7.4) served as HPLC buffer A, and HPLC-grade acetonitrile (Fisher) served as HPLC buffer B. After detritylation in 80% (v/v) acetic acid, the DNA was then desalted by a NAP-5 column (GE, Healthcare) and NAP-10 column (GE, Healthcare), quantified by UV -Vis spectrometry, and stored for future use at -20ºC. The Pb2+ substrate strand with adenosine triphosphate (rA) was purified according to the following protocols of Glen Research. After the above-mentioned detritylation, another 2′-O-triisopropylsilyloxymethyl group was removed by incubating in 100 µL of DMSO plus 125 µL of triethylamine trihydrofluoride for 2.5 h at 65°C. Then, 3 M sodium acetate (25 µL) and N-butyl alcohol (1 mL) were added and the mixture was held for at least 30 min at -20°C to precipitate DNA. The DNA pellet was dissolved with RNase free water and desalted by a NAP-5 column and NAP-10 column and stored for future use at -20°C. Preparation of Polyacrylamide DNA Conjugates. Stock solutions of 500 µM strand A and strand B were prepared in separate centrifuge tubes containing 4% acrylamide. They were placed in the vacuum desiccator at 37°C for 10 min to remove air. A solution containing 1.4% (v/v) initiator (0.05g APS dissolved in 0.5 mL of ultrapure water) and accelerator (25 µL of TEMED dissolved in 0.5 mL of ultrapure water) were then added to both stock solutions immediately. The polymerization reaction occurred in the vacuum desiccator under vacuum at 37°C for 15 min to gain linear-chain PS-A and PS-B. Unpolymerized strands and short polymers were eliminated using a 100 k NMWL ultracentrifugal filter.


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Preparation of Aptamer-crosslinked Hydrogel. PS-A (110 µM) and PS-B (110 µM) were mixed with 66 µM aptamer linker, followed by the addition of PtNPs with a final concentration of 4.6 nM in buffer for generating the cocaine hydrogel. For the Pb2+ hydrogel, 100 µM PS-A, 100 µM PS-B and 100 µM Enz were mixed with 50 µM Sublinker (Pb2+ Substrate strand), followed by the addition of PtNPs with a final concentration of 4.6 nM in buffer. For the OTA hydrogel, 110 µM PS-A and 110 µM PS-B were mixed with 70 µM aptamer linker, followed by the addition of PtNPs with a final concentration of 4.6 nM in buffer. The homogeneity of the solution was guaranteed by shaking vigorously before incubating at 65oC for 5 min in a dry bath. The aptamer crosslinked hydrogel with encapsulated PtNPs was then generated by slowly cooling to room temperature. General Experimental Procedures. When testing, 10 µL of PtNP-encapsulated hydrogel was washed three times with 100 µL of buffer. Then, 50 µL of the appropriate target was added onto the top of the gel. The reaction was allowed to continue for a certain time with gentle shaking at 150 rpm and 30°C. After that, 50 µL of supernatant was transferred to the prepared tubes and then reacted with 50 µL H2O2 for pressure measurement with a pressuremeter.

Results and Discussion Working principle of target-responsive hydrogel pressure-based assay. The basic principle of the assay is shown in Scheme 1. The target-responsive DNA hydrogel was prepared using the DNA cross-linking method reported previously.31,35,37 Briefly, polymer strands A and B (PS-A and PS-B) are firstly formed by the copolymerization of acrylamide with DNA strands A and B, respectively. Upon the addition of a linker DNA (aptamer or DNAzyme), hybridization occurs between short strands A, B and the linker DNA, which leads to the formation of the hydrogel.


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PtNP solution is premixed with the PS-A and PS-B solution so that the PtNPs can be trapped inside the 3D hydrogel network. In the presence of the target, the complex of aptamer-target forms and leads to the transfomation from gel to sol, thereby releasing the PtNPs into the supernatant. Finally, a small aliquot of the supernatant is added to H2O2 solution for O2 generation in a sealed reaction chamber where the pressure change can be sensitively readout by a handheld pressuremeter.

Scheme 1. Working principle of target-responsive hydrogel pressure-based assay. Sensitive detection of cocaine with hydrogel pressure-based assay. To prevent the abuse of highly addictive drug cocaine, simple, rapid and quantitative detection of small doses of cocaine is essential. To demonstrate the feasibility of the hydrogel pressure-based strategy, cocaineresponsive hydrogel was prepared with DNA grafted linear polyacrylamide (PS-A and PS-B) and the crosslinking DNA (cocaine aptamer), while PtNPs were trapped inside. For hydrogel stability, the optimal DNA concentrations were found to be 110 µM PS-A, 110 µM PS-B and 66 µM aptamer. The concentration of PtNPs in the hydrogel was optimized to be 4.6 nM for maximal encapsulation. First, 400 µM of cocaine was added to the prepared gel mixture. The hydrogel


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dissociated in the presence of cocaine, and thereby released the PtNPs to the supernatant after 30 min incubation. The supernatant was then transferred to H2O2 solution for O2 generation. As shown in Figure 1a, an obvious pressure change appeared in 5 min gas generation. The kinetics of PtNP release was then recorded by monitoring the pressure change during a 1-hr incubation period (Figure 1b). The pressure change was significantly increased due to the rapid release of PtNPs. By comparison, there was no obvious pressure change when there was no target. These experiments clearly suggested that the DNA hydrogel could encapsulate the PtNPs and rapidly respond to cocaine, showing the feasibility of the hydrogel pressure-based assay. Cocaine concentrations ranging from 0 to 400 µM were further tested. Figure 1c shows that the ∆P value after 5 min gas reaction was linearly proportional to the concentration of cocaine with a limit of detection (LOD) of 3.96 µM, establishing the quantitative detection capability of the hydrogel pressure-based assay. Due to the excellent stability of PtNPs, the catalytic reaction can continue for a long time with sufficient H2O2. Therefore, by prolonging the reaction time from 5 min to 15 min with saturated H2O2, higher sensitivity was achieved. As shown in Figure 1d, the hydrogel pressure-based assay with 15 min gas generation reaction can linearly detect cocaine at concentrations lower than 10 µM with a LOD of 0.12 µM. The sensitivity is one order of magnitude lower than that of commercial cocaine test kits, such as the Instant-View Cocaine Urine Dip-Strip Test (1 µM), and other POCT approaches.21,22,25,45


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Figure 1. Pressure change comparison of the hydrogel pressure-based assay after the addition of 400 µM cocaine and no cocaine (a) and their corresponding release kinetics response (b). (c) Linear standard curve for cocaine from 0 to 400 µM in 5 min gas generation. (d) Linear standard curve for cocaine from 0 to 10 µM in 15 min gas generation. Performance of hydrogel pressure-based assay in real samples. To evaluate the capability of hydrogel pressure-based assay for real sample applications, we employed it for cocaine detection in urine. The results in Figure 2a exhibited an excellent linear response between cocaine concentrations and ∆P values with a LOD of 7.16 µM within 5 min, which is comparable to the LOD for commercial cocaine test kits. Furthermore, the selectivity of the method was demonstrated. There was no obvious pressure change in the presence of cocaine metabolites (benzoylecgonine and ecgonine methyl ester) with concentrations as high as 2.5 mM. In contrast,


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250 µM cocaine could give rise to significant pressure changes (Figure 2b). These results clearly indicated that the hydrogel pressure-based assay could perform quantitative detection with excellent sensitivity and selectivity for real applications.

Figure 2. (a) Cocaine detection in human urine. Linear standard curve for cocaine from 0 to 500 µM in urine using 5 min gas generation. (b) Selectivity of hydrogel pressure-based assay for cocaine detection. To further demonstrate the accuracy of the hydrogel pressure-based assay, we compared the results of 15 different cocaine concentrations in urine acquired by hydrogel pressure-based assay with the results obtained by the traditional LC/MS method. As Figure 3 shows, compared to the gold standard method for cocaine detection, our experimental result showed a linearity of 0.985 （R2）in 15 different concentrations. These quantitation results indicated that hydrogel pressurebased assay with desired accuracy and reliability is highly applicable for real sample analysis.


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Figure 3. Correlation of the hydrogel pressure-based assay with standard LC/MS method for cocaine detection with 15 different cocaine concentrations in urine. Universality of target-responsive hydrogel pressure-based assay. Since the smart DNA hydrogel design was based on DNA hybridization, the hydrogel pressure-based assay can be further extended to a variety of targets that have specific aptamer sequences. In order to demonstrate such versatility, Pb2+ hydrogel pressure-based assay was designed based on the Pb2+-responsive DNAzyme hydrogel (Figure 4). It could quantitatively detect Pb2+ with a LOD of 3.43 nM (Figure 4a), which is much lower than the allowable limit for Pb2+ in drinking water (72 nM) set by the United States Environmental Protection Agency (EPA).46 The selectivity was demonstrated by challenging the system with high concentrations of other metal ions, such as Ni2+, Mn2+, Ca2+, Co2+, Hg2+, Fe3+, Cu2+, Zn2+, and Cd2+. The ∆P value in the presence of 1 mM control metal ions was much lower than that of 400 nM Pb2+ (Figure 4b), demonstrating the excellent selectivity of the hydrogel pressure-based assay.


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Figure 4. Design, sensitivity, and selectivity of DNAzyme-based hydrogel pressure assay for Pb2+ detection. Sequence colored in green was DNAzyme. (a) Linear standard curve for Pb2+ from 0 to 400 nM with 5 min gas generation. (b) The selectivity of hydrogel pressure assay for Pb2+ detection. Furthermore, we also designed an ochratoxin A (OTA) hydrogel pressure-based assay with the OTA aptamer (Figure 5). This OTA hydrogel pressure-based assay allowed quantification of OTA with a linear range from 0 to 2.5 µM, and the LOD was 37 nM (Figure 5a). Moreover, low concentration (2.5 µM) of target toxin could be easily discriminated from high concentration (1 mM) of other toxin molecules, such as aflatoxin M1 (AFM1), zearalenone (ZEN), T-2 toxin (T2), patulin (PAT), citrinin (CIT), Ochratoxin B (OTB) and sterigmatocystin (STC) (Figure 5b), indicating the excellent detection selectivity of the assay.


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Figure 5. Design, sensitivity, and selectivity of aptamer-based hydrogel pressure assay for OTA detection. Sequence colored in green was aptamer. (a) Linear standard curve for OTA from 0 to 2.5 µM in 5 min gas generation. (b) The selectivity of hydrogel pressure assay for OTA detection.

Conclusions In summary, a hydrogel pressure-based assay was developed by integrating a target-responsive hydrogel for selective target recognition and a pressuremeter as the signal readout for quantitative point-of-care testing. This hydrogel pressure-based strategy was demonstrated to possess several advantages, including a simple sample processing, good stability of PtNPs inside the hydrogel, and convert the target recognition event into a pressure signal. The PtNPs encapsulated inside the hydrogel with high catalytic efficiency and stability establish a quantitative and sensitive relationship between the target concentration and pressuremeter


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readout. Since the smart DNA hydrogel design is based on DNA hybridization, this method can be further extended to a variety of targets with available aptamer sequences. To verify such generality, portable and highly sensitive detection of cocain, OTA, and lead ion was achieved by the handheld pressuremeter with excellent accuracy and selectivity. The above notable advantages make this hydrogel pressure-based assay especially adaptable for portable and quantitative POCT of a broad range of targets.

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Synthesis scheme for acrylic-DMT phosphoramidite; DNA sequences used for this study. ACKNOWLEDGMENT We thank the National Science Foundation of China (21325522, 21422506, 21435004, 21275122), National Basic Research Program of China (2013CB933703), and Program for Changjiang Scholars and Innovative Research Team in University (IRT13036) for their financial support.


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Integrating Target-Responsive Hydrogel with Pressuremeter Readout

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