The development of a sensing platform with high sensitivity and specificity, especially programmability and universal applicability, for the detection of clinically relevant molecules is highly valuable for disease monitoring and confirmation but remains a challenge. Here, for the first time, we introduce the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system into an immobilization-free electrochemical biosensing platform for sensitively and specifically detecting the disease-related nucleic acids and small molecules. In this strategy, a modular rolling circle amplification (RCA) is designed to transform and amplify the target recognition event into the universal trigger DNA strand that is used as the trigger to activate the deoxyribonuclease activity of CRISPR/Cas12a for further signal amplification. The cleavage of the target-activated blocker probe allows the methylene blue-labeled reporter probes to be captured by the reduced graphene oxide-modified electrode, leading to an obviously increased electrochemical signal. We only need to simply tune the sequence for target recognition in RCA components, and this strategy can be flexibly applied to the highly sensitive and specific detection of microRNAs, Parvovirus B19 DNA, and adenosine-5′-triphosphate and the calculated limit of detection is 0.83 aM, 0.52 aM, and 0.46 pM, respectively. In addition, we construct DNA logic circuits (YES, NOT, OR, AND) of DNA inputs to experimentally demonstrate the modularity and programmability of the stimuli-responsive RCA-CRISPR/Cas12a system. This work broadens the application of the CRISPR/Cas12a system to the immobilization-free electrochemical biosensing platform and provides a new thinking for developing a robust tool for clinical diagnosis.

Development of selective and sensitive methods for on-site assay of tetracycline (TC) is of great significance for public health and food safety. Herein, a valid ratiometric fluorescence strategy using g-C 3 N 4 nanosheets coupled with Eu 3+ is designed for the assay of TC. In this strategy, both Eu 3+ and g-C 3 N 4 nanosheets serve as the recognition units of TC. The blue fluorescence of g-C 3 N 4 nanosheets can be quenched by TC via the inner filter effect (IFE); meanwhile, the red fluorescence of Eu 3+ can be enhanced by TC through the antenna effect (AE). The synergistic effect of AE and IFE caused by TC makes the developed ratiometric fluorescent sensor display a wide linear range for TC from 0.25 to 80 μM with a detection limit of 6.5 nM and a significant fluorescence color evolution from blue to red. Given its simplicity, free-label, excellent selectivity, high sensitivity, and recognizable color change, point-of-care testing systems, including smartphones and test paper-based assays, are developed for the visual sensing of TC. The integration of smartphones and test paper on a ratiometric fluorescent sensor greatly reduces the detection cost and time, providing a promising method for the qualitative discernment and semi-quantitative assay of TC on-site. Moreover, the potential application of the approach is also verified by detecting TC in milk.

In this work, on the basis of a new 2D DNA nanoprobe (DNP) and an enzyme-free-target-recycling amplification, an electrochemical biosensor is developed for the ultrasensitive detection of microRNA-21 (miRNA-21). Herein, two ferrocene-labeled bipedal DNPs, which show small steric hindrance and strong stability, are prepared on the basis of the mechanism of the proximity-ligation assay (PLA), improving the space utilization. In the presence of the target, miRNA-21, and a hairpin DNA strand, the DNP will collapse, and then two ferrocene-labeled DNA strands and the miRNA-21 will be simultaneously released from the electrode surface through toehold-mediated strand-displacement reactions (TSDRs), leading to a decrease in the electrochemical signal and realization of enzyme-free target recycling. As a result, the one input target, miRNA-21, could release 2 N ferrocene-labeled DNA strands, achieving a dramatic decrease in the electrochemical signal. Combining DNPs and enzyme-free target recycling, this proposed biosensor showed a linear dependence with miRNA-21 concentration, ranging from 1.0 fM to 10 nM with a detection limit of 0.31 fM. In addition, it is worth mentioning that this biosensor can be regenerated through incubating with three assistant-DNA strands, realizing the reuse of raw materials. Surprisingly, the elaborated biosensor provides a novel strategy for building controllable DNA nanoprobes for the sensitive detection of various biomarkers.

Herein, for the first time, we engineered click chemistry reaction to trigger a 3D DNA walking machine for ultrasensitive electrochemical detection of copper ion (Cu), which provided a convenient access to overcome the shortcomings of poor selectivity and limited amplification efficiency in traditional determination of Cu. Click chemistry reaction drove azido-S2 to bind with alkynyl-S1 for the formation of a walker probe on aminated magnetic polystyrene microsphere@gold nanoparticles (PSC@Au), which opened the hairpin-locked DNAzyme. In the presence of magnesium ion (Mg), the unlocked DNAzyme was activated to cleave the self-strand at the facing ribonucleotide site, accompanied by the release of product DNA (S3) and the walker probe. Therefore, the walker probe was able to open the adjacent hairpin-locked DNAzyme strand and then be released by DNAzyme cleavage along the PSC@Au-DNAzyme track. Eventually, the liberated single-strand S3 induced catalytic hairpin assembly (CHA) recycling, resulting in the capture of a large number of methylene blue-tagged hairpin DNA (MB-H2) on the sensor surface and significant electrochemical responses. By coupling click chemistry reaction with the dual-amplification strategy of the 3D DNA walking machine and CHA recycling, the proposed biosensor not only demonstrated high accuracy and selectivity for Cu detection in real samples but also showed excellent performance for Cu detection with a wide linear range of 1.0 pM to 500 nM and low detection limit of 0.33 pM. Moreover, this elaborated biosensor could be readily expanded to Mg detection with a constant concentration of Cu, which paves a new way to apply the 3D DNA walking machine in various ion sensings.