Novel Biosensing Methods For Instrument-free Quantitative Detection

Liquid-phase colorimetric analysis(LPCA)usually uses organic dyes,enzyme substrates,and noble metal nanoparticles as colorimetric probes to convert detection events into changes in color intensity.The most existing LPCAs rely on the one-dimensional(1D)information of color intensity in the final reaction solution,which we refer to as 1D LPCA.The 1D LPCA has long been the favor of analysts for the outstanding advantages of simple operation,cost-low,visual qualitative or semi-quantitative detection.The fly in the ointment is the need to use UV-Vis spectrophotometer and other optical instruments to achieve quantitative measurements of analytes.In this thesis,we have developed two new methods of instrument-free quantitative biosensing with original innovation by using cysteamine,adenosine and heavy metal ions as model analytes and combined with two-dimensional(2D)LPCA strategy.(1)It seems impossible for existing gold nanoparticle-based liquid-phase colorimetric assays(GNP-LPCAs)that generally rely on one dimensional(1D)information of color type/intensity of the GNP solution to achieve quantitative detection without the use of UV-Vis spectrometers.In Chapter 2,we attempt to address this issue by describing a new class of GNP-LPCA termed as two dimensional(2D)GNP-LPCA based on cysteamine-mediated aggregation of GNPs.Unlike the 1D GNP-LPCAs in which the aggregated GNPs would make the whole color of the homogeneous solution change from red to blue,the 2D GNP-LPCA uses the formamide as an “intelligent adjuster” to produce a top-bottom double-color(red-blue or blue-red)solution.More importanly,the color length is proportional to the cysteamine concentration and thus can be explored as the additional(secondary)dimensional information for equipment-free quantitative analysis.The experimental results show that under the optimal conditions,the linear range of cysteamine obtained is 1.3 ~ 80 ?M,and the detection limit by the naked eye is 1.3 ?M.The utility and expansibility of the 2D GNP-LPCA is well demonstrated with the design of an aptamer-based 2D GNP-LPCA for for simple,low-cost,sensitive and specific detection of ademosine as a model analyte in buffer and human serum samples by the naked eye.The experimental results show that under the optimal conditions,the linear range of adenosine obtained is from 9.7 nM to 10 mM,with a visual detection limit of 9.7 nM.The recovery results of assaying adenosine in human serum samples are also satisfactory.(2)Silver is one of the most toxic heavy metals.Most of the current quantitative detection methods for silver ions(Ag+)typically use large and expensive analytical equipments.In Chapter 3,we describe a new method for visual quantitative point-of-care testing(POCT)of Ag+ based on the two-dimensional liquid-phase colorimetric assay strategy without using any analytical instruments.In this equipment-free method,formamide is used to dissolve 3,3',5,5'-tetramethylbenzidine(TMB)probe.When the Ag+ aqueous sample solution is dropped into such solution,the TMB will be rapidly oxidized by the ion analyte to produce blue quinoid benzidine.Since the product is more likely to be formed in the aqueous phase,a unique top-bottom blue-colorless two-color mixture solution is finally obtained.The ‘‘smart'' formamide solvent is able to adjust the blue color length as the analyte level varies.That is,with the increase of Ag+ concentration,the length of the blue color in the solution increases in the final two-color mixture solution.The experimental results show that under the optimal conditions,the linear range of Ag+ obtained is from 62.5 ?M to 4 mM,with a visual detection limit of 24.9 ?M.The recovery results of assaying pond water samples are also satisfactory.