Aptamer-based Dry-reagent Strip Biosensor for

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Aptamer-based Dry-reagent Strip Biosensor for Detection of Small Molecule ATP. Qiang Zhang,*1 Hongbin Qiu,1 Fangqiang Tang,1 Ye Tao,1 Baosheng Guan,1 ...
Aptamer-based Dry-reagent Strip Biosensor for Detection of Small Molecule ATP Qiang Zhang,*1 Hongbin Qiu,1 Fangqiang Tang,1 Ye Tao,1 Baosheng Guan,1 Xuechen Li,2 and Wei Yang3 1 School of Public Health, Jiamusi University, 154007 Jiamusi, P. R. China 2 University Hospital, Jiamusi University, 154007 Jiamusi, P. R. China 3 Central Hospital of Jiamusi City, 154002 Jiamusi, P. R. China (E-mail: [email protected])

A novel lateral-flow strip biosensor was developed for high specificity, low-cost and visual detection of small molecule ATP by using anti-ATP aptamer-functionalized gold nanoparticles (AuNPs) as sensing elements.

REPRINTED FROM

Vol.45 No.3

2016 p.289–290 CMLTAG March 5, 2016

The Chemical Society of Japan

Received: November 19, 2015 | Accepted: December 28, 2015 | Web Released: January 8, 2016

CL-151077

Aptamer-based Dry-reagent Strip Biosensor for Detection of Small Molecule ATP Qiang Zhang,*1 Hongbin Qiu,1 Fangqiang Tang,1 Ye Tao,1 Baosheng Guan,1 Xuechen Li,2 and Wei Yang3 1 School of Public Health, Jiamusi University, 154007 Jiamusi, P. R. China 2 University Hospital, Jiamusi University, 154007 Jiamusi, P. R. China 3 Central Hospital of Jiamusi City, 154002 Jiamusi, P. R. China (E-mail: [email protected]) A novel dry-reagent strip biosensor was developed for high specificity, low-cost, and visual detection of small molecule ATP by using anti-ATP aptamer-functionalized gold nanoparticles (AuNPs) as sensing elements. Keywords: Dry-reagent strip biosensor | Aptamer-functionalized Au nanoparticle | Adenosine triphosphate (ATP)

As an important substrate in living organisms, adenosine triphosphate (ATP) plays a critical role in the regulation of cellular metabolism and biochemical pathways in cell physiology.1 ATP detection not only can be used to indicate several diseases,2 such as infection and inflammation of the urinary tract, Alzheimer’s disease, neonatal hypoxia, and hepatic disorders, but also can be used to control biological treatment reactors, guide biocide dosing programs, determine drinking water cleanliness, manage fermentation processes, assess soil activity, and measure equipment or product sanitation. Therefore, the determination of ATP is essential in clinic diagnosis as well as environmental health. Several commonly used techniques for ATP detection, such as HPLC, chemiluminescence, fluorescence, mass spectrometry, enzymatic assays, and electrochemistry, are usually time-consuming, lacking selectivity, or needing complicated instruments.1,3 Thus, development of a simple, fast, and selective ATP detection method remains a great challenge. Recently, a lateral flow biosensor, also called a dry-reagent strip biosensor, is enjoying great popularity because they are inexpensive, rapid, and portable.4 Currently, most of the strip biosensors that have been extensively applied are based on the use of antibodies as affinity probes. However, the utilization of antibodies may encounter some drawbacks with their production and modification, and searching for other alternative candidates is ongoing. Aptamers, selected from combinatorial libraries using systemic evolution of ligands by exponential enrichment (SELEX), are usually single-strand DNAs or RNAs, which can bind with high affinity and specificity to a wide range of targets including small molecules.5 In spite of the similar identification principle for targets with antibodies, aptamers can provide several advantages over antibodies such as simple synthesis, easy labeling, good stability, wide applicability, and high sensitivity.6 At present, some aptamer-based strip biosensors have been established for the detection of cells and macromolecules, such as proteins and nucleic acids.7 However, this method for the detection of small molecule ATP has not been developed. In the present study, we for the first time report a strip biosensor for ATP analysis by combining the high selectivity and affinity of aptamers with the unique optical properties of Au nanoparticles (AuNPs). A schematic diagram of strip fabrication is shown in Figure 1. The biosensor consists of the following components: a nitrocellulose membrane, a conjugate pad, a sample pad, and an

Chem. Lett. 2016, 45, 289–290 | doi:10.1246/cl.151077

Figure 1. The fabrication (a) and measurement principle (b) of the aptamer-based dry-reagent strip.

absorption pad. On the nitrocellulose membrane (2.5 © 20 cm), the test line and control line were prepared by immobilizing the streptavidin-conjugated test probe (5¤-TACTCCCCCAGGTAAbiotin-3¤, complementary with part of the anti-ATP aptamer) and streptavidin-conjugated control probe (5¤-biotin-AAAAAAAAAAAAA-3¤), respectively, according to the reported method.7a The distance between two lines was about 0.5 cm. The membrane was dried at room temperature for 1 h and stored in a desiccator at 4 °C. The conjugate pad was prepared by spraying AuNPs­aptamer (5¤-SH-(CH2)6-TTTTTTTTTTTTTACCTGGGGGAGTATTGCGGAGGAAGGT-3¤) conjugate solution on the glass fiber (1.0 © 20 cm). AuNPs­aptamer conjugates were synthesized via the wellknown gold­sulfur chemistry. Firstly, AuNPs (13 « 1 nm in diameter) were synthesized according to the citrate reduction of H[AuCl4].8 The concentration of the prepared AuNPs was about 12 nM, calculated by the quantity of the starting material (H[AuCl4]) and the size of AuNPs at the wavelength of 519 nm.9 Next, 1 mL of AuNPs solution was mixed separately with 200 ¯L of thiol-aptamer (12 ¯M) to obtain a final concentration of 10 nM AuNPs and 2 ¯M oligonucleotides and reacted at room temperature for 24 h. The mixtures were centrifuged for 25 min at 15000 rpm to remove the excess thiol-aptamer. The sample pad (1.5 © 20 cm) was saturated with TBS (20 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.2). Then, it was dried and stored in a desiccator at room temperature. The absorption pad (1.6 © 20 cm) was used without treatment. All of the parts were laminated 0.2 cm with each other in sequence and pasted onto the

© 2016 The Chemical Society of Japan | 289

plastic back plate. The strips with 6.0 cm in length and 0.4 cm width were cut. The principle of the developed dry-reagent strip measurement is based on the competitive reaction between the test probe (test line) and the target ATP to combine with aptamers, and the protocol is illustrated in Figure 1b. In a typical assay, the sample solution containing ATP was applied on the sample application pad. Subsequently, the solution migrated by capillary action and rehydrated the AuNPs­aptamer conjugates. Then, the binding between ATP and AuNPs­aptamer occurred, and decreased the binding of AuNPs­aptamer to test probe in the test line and caused the red color intensity to become weaker. The more target ATP in the solution, the weaker intensity in the test line was shown. Once the solution passed through the control zone, the excess AuNPs­ aptamer was captured by the control probe in the control line, thus forming a second red band. In the absence of ATP, a red band is observed in the test line. In this case, a red control band (control line) shows that the aptamer-based dry-reagent strip is working properly. The color changes of the test line are observed by eye and relative changes of optical intensity on the test line (compared with 0 ¯M of ATP or 100 ¯M of GTP) are analyzed by Image J software. The sensitivity of the aptamer-based dry-reagent strip was determined by testing various concentrations of ATP standard samples (0­500 ¯M in TBS). The visual detection limit of the assay could be defined as the minimum ATP concentration producing the color of the test line significantly weaker than that of control line for 10 min. As shown in Figure 2a, the intensity of the red color on the control line was nearly the same on all strips, which showed the validity of strip detection. Also, with an increase in target ATP in the detection solution, the red color intensity on the test line decreased. The color density of the test line showed visual weaker than that of the control line when the ATP concentration was 20 ¯M. According to the definition of the qualitative sensitivity of the strip detection, 20 ¯M could be treated as the visual detection limit of the aptamer-based strip. The color density of the test is proportional to ATP concentration in the range 20­500 ¯M of ATP. In principle, the fabricated strip should be very specific due to the high recognition ability of the aptamer to ATP. Nevertheless, we investigated the specificity of the aptamer-based strip using three different nucleotides, GTP, CTP, and UTP, to replace ATP in the samples. The results are presented in Figure 2b. A high response was observed when 100 ¯M ATP was tested, whereas negligible signals were obtained in presence of 100 ¯M of GTP, CTP, or UTP, indicating good specificity of the fabricated strip. In order to test the practical applicability of the aptamer-based strip biosensor, experiments were performed by the detection of

Figure 2. Sensitivity assay (a), specificity assay (b), and real sample assay (c) of the aptamer-based dry-reagent strip for ATP. C: control line; T: test line; 1­7: the concentrations of ATP were 0, 10, 20, 50, 100, 200, 500 ¯M; 8­11: GTP, CTP, UTP, ATP; 12­14: the concentrations of ATP in urine were 0, 20, 100 ¯M. The intensities of test line were analyzed by Image J.

290 | Chem. Lett. 2016, 45, 289–290 | doi:10.1246/cl.151077

ATP in a real sample. Simulated samples were prepared by spiking a known amount of ATP into urine. The results are shown in Figure 2c. It can be seen that the detection limit of ATP in the simulated urine was 20 ¯M, which is the same as the detection limit of ATP in TBS. This result indicated that the developed aptamerbased strip biosensor was feasible to detect ATP in real samples. In conclusion, we have successfully developed an aptamerbased strip sensor for visual detection of ATP. Although the sensitivities of the assays described here were lower than some instrument-based assays. However clearly, it is easy, rapid, and convenient to perform and does not need sophisticated equipment compared to those techniques, and could be accomplished within 10 min without complicated handling procedures. With respect to its overall speed and simplicity, the aptamer-based dry-reagent strip could be a potential alternative tool for rapid and sensitive on-site screen and detection of ATP. This work was financially supported by the National Natural Science Foundation of China (Nos. 31101250 and 81273174), Natural Science Foundation of Heilongjiang Province of China (No. H201372), and Supporting Plan Project for Youth Academic Backbone of General Colleges and Universities of Heilongjiang Province (No. 1254G057), and Key Project of Jiamusi University (No. Sz2011-006). References 1 T. Pérez-Ruiz, C. Martínez-Lozano, V. Tomás, J. Martín, Anal. Bioanal. Chem. 2003, 377, 189. 2 a) C. Zhang, R. A. Rissman, J. Feng, J. Alzheimer’s Dis. 2015, 44, 375. b) C. Auger, A. Alhasawi, M. Contavadoo, V. D. Appanna, Front. Cell Dev. Biol. 2015, 3, 40. c) K. Gill, H. Horsley, A. S. Kupelian, G. Baio, M. De Iorio, S. Sathiananamoorthy, R. Khasriya, J. L. Rohn, S. S. Wildman, J. Malone-Lee, BMC Urol. 2015, 15, 7. d) F. A. Mateos, J. G. Puig, T. H. Ramos, R. H. Carranza, M. E. Miranda, R. C. Gasalla, in Purine and Pyrimidine Metabolism in Man VI: Part A: Clinical and Molecular Biology in Advances in Experimental Medicine and Biology, Springer, 1989, Vol. 253A, p. 345. doi:10.1007/ 978-1-4684-5673-8_56. 3 a) L. Mora, A. S. Hernández-Cázares, M.-C. Aristoy, F. Toldrá, Food Chem. 2010, 123, 1282. b) R. Corriden, P. A. Insel, W. G. Junger, Am. J. Physiol.: Cell Physiol. 2007, 293, C1420. c) G. Davis, M. J. Green, H. A. O. Hill, Enzyme Microb. Technol. 1986, 8, 349. d) Y.-F. Huang, H.-T. Chang, Anal. Chem. 2007, 79, 4852. e) A. Ishida, Y. Yamada, T. Kamidate, Anal. Bioanal. Chem. 2008, 392, 987. 4 D. Koizumi, K. Shirota, R. Akita, H. Oda, H. Akiyama, Food Chem. 2014, 150, 348. 5 a) R. Knight, M. Yarus, RNA 2003, 9, 218. b) D. S. Wilson, J. W. Szostak, Annu. Rev. Biochem. 1999, 68, 611. 6 a) C. Reinemann, B. Strehlitz, Swiss Med. Wkly. 2014, 144, w13908. b) P. Majumder, K. N. Gomes, H. Ulrich, Expert Opin. Ther. Pat. 2009, 19, 1603. 7 a) G. Liu, X. Mao, J. A. Phillips, H. Xu, W. Tan, L. Zeng, Anal. Chem. 2009, 81, 10013. b) H. Xu, X. Mao, Q. Zeng, S. Wang, A.-N. Kawde, G. Liu, Anal. Chem. 2009, 81, 669. c) X. Mao, Y. Ma, A. Zhang, L. Zhang, L. Zeng, G. Liu, Anal. Chem. 2009, 81, 1660. 8 J. Wang, A. Munir, Z. Li, H. S. Zhou, Biosens. Bioelectron. 2009, 25, 124. 9 R. C. Mucic, J. J. Storhoff, C. A. Mirkin, R. L. Letsinger, J. Am. Chem. Soc. 1998, 120, 12674.

© 2016 The Chemical Society of Japan