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Gold Nanostar Enhanced Surface Plasmon Resonance Detection of an Antibiotic at Attomolar Concentrations via an Aptamer-Antibody Sandwich Assay Suhee Kim, and Hye Jin Lee Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 18 May 2017 Downloaded from http://pubs.acs.org on May 18, 2017
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Analytical Chemistry
Gold Nanostar Enhanced Surface Plasmon Resonance Detection of an Antibiotic at Attomolar Concentrations via an Aptamer-Antibody Sandwich Assay
Suhee Kim and Hye Jin Lee*
Department of Chemistry and Green-Nano Materials Research Center, Kyungpook National University, 80 Daehakro, Buk-gu, Daegu-city, 41566, Republic of Korea
*Corresponding author: E-mail address:
[email protected]; Tel. + 82 053 950 5336; Fax +82 053 950 6330; Orcid ID 0000-0002-2181-6813; Postal address: Department of Chemistry and Green-Nano Materials Research Center, Kyungpook National University, 80 Daehakro, Buk-gu, Daegu-city, 41566, Republic of Korea
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Analytical Chemistry
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Abstract A new sandwich assay for tetracycline (TC) involving a DNA aptamer and antibody pair is demonstrated in conjunction with gold nanostar (GNS) enhanced surface plasmon resonance (SPR) to achieve detection in the low attomolar range. GNS particles were covalently functionalized with the antibody probe (antiTC) and integrated into a surface sandwich assay in conjunction with a SPR gold chip modified with the TC-specific aptamer. After it was demonstrated that both affinity probes can bind simultaneously to TC, optimization of the assay was performed using either antiTC only or GNS-antiTC conjugates to interact with aptamer/TC complexes present on the chip surface. Target concentrations as low as 10 aM could be detected using GNS-antiTC’s, which was >103 times greater in performance than when using antiTC only. In addition, good selectivity was achieved with respect to other tetracycline derivative antibiotics such as oxytetracycline (OTC) and chlorotetracycline (CTC) both which are structurally similar to TC. As a demonstration of trace antibiotic analysis in environmental samples, the GNS enhanced sandwich assay was applied to analyze TC added to aliquots of local river water and the results validated by comparing to conventional HPLC analysis.
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Analytical Chemistry
Introduction
The prevalent use of pharmaceutical products such as antibiotics have led to increasing concerns regarding environmental contamination and the potential impact of this on human and animal health as well as contributing to increasing bacterial drug-resistance.1-3 For example, tetracyclines (TC’s) including tetracycline (TC) itself alongside a number of analogues such as oxytetracyline (OTC) and chlortetracycline (CTC), are used in the treatment of a wide range of infections and diseases.4,5 Residues of TC’s have been reported found in meat6, milk7 and eggs8 which is a significant contributing factor to the promotion of antibiotic resistant microorganisms. In addition to food products5-8, TC’s and also specific genes in species associated with TC-resistance have been reported in ground water and other wastewater lagoon types.9-12 Particularly challenging for environmental analysis, is that pharmaceutical residues such as TC’s are present at very low levels making them very difficult to detect directly unless there is a build-up of concentration such as in organisms.9 It is thus important to develop analytical methods that can monitor residual antibiotics in aqua environments at extremely low levels.13,14 A wide variety of detection methodologies for TC’s have been reported8,10,12,15-21 including chromatographic methods8, mass spectrometry12,16,18, enzyme linked immunosorbent assay (ELISA)19,20, and amperometric immunosensors21 with typical detection performance ranging from 0.01 to 10 g/L.
Surface plasmon resonance (SPR) is now a well-established technique which offers direct detection and real-time monitoring advantages22-25 and has been applied for the analysis of antimicrobial residues in aqua environmental analysis.5,22,26 However, as SPR is based on measuring changes in refractive index, achieving suitable specificity and sensitivity becomes 3
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more challenging for the detection of smaller sized drug molecules. For example, Scholz et al reported the SPR detection method of TC in milk sample solutions.24 The label-free detection of a small molecule (MW
