Research Of Signal Amplification Strategies For Scanning Electrochemical Microscopy (SECM)-based Biosensing Platforms

Scanning electrochemical microscopy (SECM) was introduced by Bard in 1989. It involves current measurements through an ultramicroelectrode (UME) scanning over a substrate in a solution. It is now being applied to the study of biological processes and immobilized biomolecules, such as gene, enzyme, peptide, antigen and antibody. Compared with conventional electrochemical methods, the SG/TC mode of SECM realizes the separation of biomolecules supporter and electrochemical signal reporter. In other words, the electrochemical signal is obtained by the SECM tip, while both the target biomolecules and signal amplification elements are immobilized on the substrate. Therefore, the conductivity of the substrate cannot affect the tip signal current and the electrochemical reaction characteristics.In this paper, we focused on exploring the SECM specific signal amplification strategies to improve the sensitivity of biomolecules detection, mainly containing the following two aspects. (1) Enhanced by the long self-assembled DNA concatemers, which was restricted as a signal amplification element in a conventional electrochemical method, an ultrasensitive SECM-based DNA biosensing platform was fabricated. This signal amplification strategy was also used for the SECM-based DNA microarray biosensing platform, realizing the simultaneous detection of multiple target DNA molecules with high sensitivity. This scheme holds great potential for genome sequencing and other high-throughput analyzing. (2) The SECM tip was modified, in order to be not only used as a simple interface for obtaining electrochemical signal, but also used to further generate catalytic amplification.This paper consists of four chapters as follows:Chapter 1:IntroductionIn this chapter, we briefly introduced the SECM, including its background, basic structure, working mode, and its current research status in the three areas of DNA analysis, immunoassays and tip chemical modification methods. Finally, we illustrated the main research contents in this paper and their significance.Chapter 2:An ultrasensitive SECM-based DNA biosensing platform amplified with long self-assembled DNA concatemersThe long DNA concatemers have been well developed to fabricate various biosensing platforms for the signal amplification. Herein, this signal amplification strategy was firstly used for an ultrasensitive SECM-based DNA biosensing platform. This platform was constructed through the hybridization of target DNA (TD) with thiol-tethered DNA capture probes (CP), immobilized on the gold substrate surface, and biotinylated DNA signal probes (SP), which formed then the long DNA concatemers through the continuous self-assembly with alternating DNA auxiliary probes (AP). The streptavidin-horseradish peroxidase (HRP) was linked to the long DNA concatemers through biotin-streptavidin interaction. In the HRP-catalyzed reaction, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) with H2O2 at the modified substrate surface where sequence-specific hybridization had occurred, and the BQ generated could be monitored by a SECM tip. This platform exhibited a relatively low detection limit of 0.18 aM as estimated by the 3a rule.Chapter 3:An ultrasensitive SECM-based DNA biosensing platform amplified with long self-assembled DNA concatemersAs long self-assembled DNA concatemers exhibited a significant amplification effect on SECM-based biosensing platforms, we fabricated a SECM-based DNA microarray biosensing platform using this signal amplification strategy in this chapter. The DNA microarray was constructed by the robot through immobilizing the DNA probes on the amino modified glass substrate. The fabrication process of DNA microarray biosensing platform was similar to that in Chapter 2. In the HRP-catalyzed reaction, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) with H2O2 at the modified substrate surface where sequence-specific hybridization had occurred, and the BQ generated could be monitored by a SECM tip. Therefore, the images of the substrate at different sites could be obtained. Four kinds of TDs (100 fM) as the models were detected simultaneously by using this proposed strategy, which also demonstrated sufficient selectivity to distinguish specific DNA sequences and good reproducibility. This method opens a promising direction to improve the SECM sensitivity for high-throughput DNA detection.Chapter 4:SECM-based glucose biosensing platform enhanced by Prussian Blue (PB) film modified Pt tipIn this chapter, a layer of Prussian Blue was modified by electrodeposition on the Pt tip, and the glucose biosensing platform was fabricated with the glucose oxidase (GOD) as the model enzyme. During the experiment, PB film modified Pt tip exhibited excellent stability and a good electrocatalytic activity to H2O2. Served as a signal amplification element, PB film greatly improves the detection sensitivity. This scheme holds great potential for improving the sensitivity of various SECM-based biosensing platforms enhanced by PB film modified tip.