An Integrated Multi‐Function Heterogeneous Biochemical Circuit for High‐Resolution Electrochemistry‐Based Genetic Analysis

Abstract

Modular construction of an autonomous and programmable multi-functional heterogeneous biochemical circuit that can identify,t ransform, translate,a nd amplify biological signals into physicochemical signals based on logic design principles can be apowerful means for the development of av ariety of biotechnologies.T oe xplore the conceptual validity,w ed esign aC RISPR-array-mediated primer-exchange-reaction-based biochemical circuit cascade,w hich probes as pecific biomolecular input, transform the input into as tructurally accessible form for circuit wiring,t ranslate the input information into an arbitrary sequence,a nd finally amplify the prescribed sequence through autonomous formation of as ignaling concatemer.T his upstream biochemical circuit is further wired with ad ownstream electrochemical interface,d elivering an integrated bioanalytical platform. We program this platform to directly analyzethe genome of SARS-CoV-2 in human cell lysate,d emonstrating the capability and the utility of this unique integrated system.

Clustered Regularly Interspaced short palindromic repeats‐Based Microfluidic System in Infectious Diseases Diagnosis: Current Status, Challenges, and Perspectives

“…[149] Recently, a series of electronic CRISPR-based microfluidic devices has been developed for infectious diseases diagnosis. [96][97][98]112,113,126,127,148] For example, a CRISPR-arraymediated primer-exchange-reaction (PER)-based biochemical circuit cascade was designed based on the effect of signaling cascade amplification to detect the SARS-CoV-2 genome with the nanomolar (nM)-level sensitivity in 90 min. [98] This surface-modified electronic sensor in a microfluidic device was shown to be stable during long-term dry storage.…”

“…Finally, the amplification process is nonlinear, limiting the quantitative detection of infectious diseases. [ 45 , 94 ] To overcome these limitations, various amplification‐free CRISPR‐based methods have been explored, such as multiple crRNA‐based determination, [ 21 , 95 ] signaling cascade amplification, [ 96 , 97 , 98 ] and digital technology. [ 20 , 35 , 61 , 63 , 92 , 99 , 100 ] It has been known that the CRISPR‐based amplification‐free digital RNA detection (SATORI) was designed with multiple crRNA strategies and CRISPR/Cas13a cleavage.…”

“…Furthermore, the signal cascade strategy was utilized in the CRISPR‐array‐mediated primer‐exchange‐reaction‐based biochemical circuit cascade, in which an autonomous signal converter was designed to link the upstream biochemical circuit with the downstream electrochemical readout (Figure 4B ). [ 98 ]…”

“…[ 96 , 97 , 98 , 112 , 113 , 126 , 127 , 148 ] For example, a CRISPR‐array‐mediated primer‐exchange‐reaction (PER)‐based biochemical circuit cascade was designed based on the effect of signaling cascade amplification to detect the SARS‐CoV‐2 genome with the nanomolar (nM)‐level sensitivity in 90 min. [ 98 ] This surface‐modified electronic sensor in a microfluidic device was shown to be stable during long‐term dry storage. [ 126 ] Another CRISPR/Cas12a‐based electrochemical biosensor (E‐CRISPR) was designed to diagnose HPV‐16 and parvovirus B19 with picomolar (pM) sensitivity.…”

Research progress of CRISPR-based biosensors and bioassays for molecular diagnosis

“…Liu et al explored a primer-exchange-reaction (PER)-based biochemical circuit cascade using a Cas9 enzyme with only single-stranded cleavage activity. Driving a pair with different sgRNAs of Cas9 generates dsDNA with sticky ends, ligated to a hairpin-structure substrate, which subsequently generates a large number of short fragments that report the electrical signal of the detected DNA (Dai et al, 2020). Huang's team developed a dCas9-ECL probe for sensitive and single-base specific DNA detection with good resistance to nonspecific interference and a low false positive detection rate (Wu L. et al, 2022).…”