This article provides a comprehensive review of biosensing with DNAzymes, providing an overview of different sensing applications while highlighting major progress and seminal contributions to the field of portable biosensor devices and point-of-care diagnostics. Specifically, the field of functional nucleic acids is introduced, with a specific focus on DNAzymes. The incorporation of DNAzymes into bioassays is then described, followed by a detailed overview of recent advances in the development of in vivo sensing platforms and portable sensors incorporating DNAzymes for molecular recognition. Finally, a critical perspective on the field, and a summary of where DNAzyme-based devices may make the biggest impact are provided.

Biosensing with DNAzymes

Nucleic acids are natural biopolymers of nucleotides that store, encode, transmit and express genetic information, which play central roles in diverse cellular events and diseases in living things. The analysis of nucleic acids and nucleic acids-based analysis have been widely applied in biological studies, clinical diagnosis, environmental analysis, food safety and forensic analysis. During the past decades, the field of nucleic acids analysis has been rapidly advancing with many technological breakthroughs. In this review, we focus on the methods developed for analyzing nucleic acids, nucleic acids-based analysis, device for nucleic acids analysis, and applications of nucleic acids analysis. The representative strategies for the development of new nucleic acids analysis in this field are summarized, and key advantages and possible limitations are discussed. Finally, a brief perspective on existing challenges and further research development is provided.

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Nucleic Acids Analysis

Forged signature threatens the authenticity of personal identity. Here, an effective SERS anti-counterfeiting system is designed for personal signatures. Mixed ligands improve the complexity of Raman spectra and expand the coding capacity. Fourteen distinct combinations are created from mere five ligands, and great expansion is possible with modest expansion of the ligand library. On the other hand, the (Au-aggregate)@Ag@PSPAA nanostructure significantly increases the surface-enhanced Raman scattering (SERS) intensity and stability so that excellent performance is achieved in SERS detection. By integrating these strategies, SERS inks are produced and applied in signature anti-counterfeiting. The resulting spectra are converted to barcodes that are readily detected through a smart phone APP. With these improvements, this work brings SERS one step closer toward practical applications in signature anti-counterfeiting.

Multiplexed SERS Barcodes for Anti-Counterfeiting.

Sensitive, fast and reliable detection of UO22+ ions is of great significance in nuclear industry and environment protection, due to the serious threats of UO22+ ions to human health. However, such suitable sensor is still rare. Herein, an ultrasensitive and recyclable SERS-microfluidic biosensor with specific UO22+ response has been developed. Aptamer-modified ZnO-Ag hybrids arrays was firstly designed and utilized as highly functional sensor by colloidal crystals templating method. The relationship between aptamers (different types, length and reaction time) and UO22+ ions was fully screened to improve the detection efficiency. In the absence of UO22+, Rhodamine B (RhB)-labeled double-stranded DNA formed a rigid structure, and weak Raman signals were detected. After pumping the UO22+ solution into microdevice, DNAzyme-cleavage reaction was triggered. And RhB-modified 5′-single DNA strand (cleavage production) dropped down to the surface of SERS substrates, leading to strong Raman signals. After signal amplification, the detection limit of UO22+ achieved as low as 7.2 × 10−13 M, which is nearly five orders below the EPA-defined maximum contaminant level. More importantly, the specially designed microfluidic device could be reused and refreshed by supplementation of substrate strands DNA for three times. A further application to determine UO22+ ions in natural real systems such as tap water and river water was also complied with good recoveries and RSDs. This SERS-based microfluidic sensor shows great potential for in-the-field sensing platforms, due to its ultra-sensitivity, high efficiency, and portability.

Ultrasensitive, recyclable and portable microfluidic surface-enhanced Raman scattering (SERS) biosensor for uranyl ions detection

Applications of surface-enhanced Raman spectroscopy in detection fields.

The uranyl ion (UO22+) poses high risks to human health and the environment, hence its detection and monitoring is of utmost significance. However, the development of an ultra-sensitive, high-efficiency and convenient approach for on-site detection of UO22+ remains a challenge. Herein, a reliable and reusable surface-enhanced Raman spectroscopy (SERS)-based microfluidic biosensor was developed for rapid detection of UO22+ in real samples. The detection protocol involved the reaction of 5′-Rhodamine B (RhB)-labeled double-stranded DNA for UO22+-specific DNAzyme-cleavage reaction in a U-shaped micro-channel. Then, the reaction products were delivered into three parallel samples for high-throughput tests by SERS biochips, where 3D ZnO-Ag mesoporous nanosheet arrays (MNSs) were modified with a single-stranded DNA (ssDNA). The ssDNA was sequence-complementary with the 5′-RhB-labeled cleaved-stranded DNA (csDNA) from the reaction products. By the hybridization of ssDNA and csDNA, the signal probe RhB was fixed close to the surface of the ZnO-Ag MNSs to enhance the Raman signal. The limit of detection for UO22+ with the microfluidic-SERS biosensor was 3.71× 10−15 M. An over 20,000-fold selectivity towards UO22+ response was also achieved in the presence of 15 other metal ions. The high-throughput microfluidic-SERS biosensor operated well for practical UO22+ detection, with excellent recoveries in contaminated river and tap water from 95.2% to 106.3% (relative standard deviation (RSD) n =6). Although the SERS-based microfluidic biosensor developed in this study was deployed for the detection of UO22+, the reusable and high-efficiency system may be expanded to the detection of other analytes on-site.

Yu Liu

Papers

Ultra-sensitive detection of uranyl ions with a specially designed high-efficiency SERS-based microfluidic device