The clustered regularly interspaced short palindromic repeats (CRISPR) technology has been widely applied for nucleic acid detection because of its high specificity. By using the highly specific and irreversible bond between HaloTag and its alkane chlorine ligand, we modified dCas9 (deactivated CRISPR/Cas9) with biotin as a biosensor to detect nucleic acids. The CRISPR biosensor was facilely prepared to adequately maintain its DNA‐recognition capability. Furthermore, by coupling biolayer interferometry (BLI) with the CRISPR biosensor, a real‐time, sensitive, and rapid digital system called CRISPR‐BLI was established for the detection of double‐stranded DNA. The CRISPR biosensor immobilised on the biolayer could recruit the target DNA onto the biosensor surface and change its optical thickness, resulting in a shift in the interference pattern and responding signal of the BLI. The CRISPR‐BLI system was further applied to detect the ALP gene of Escherichia coli DH5α combined with a polymerase chain reaction, which demonstrated a linear range from 20 to 20 000 pg and a low detection limit (1.34 pg). The CRISPR‐BLI system is a promising approach for rapid and sensitive detection of target DNA analytes.

(1) Background: Chemiluminescent enzyme immunoassay (CLEIA) is an efficient analytical method. Alkaline phosphatase (ALP) with high specific activity is the basis for CLEIA to achieve high sensitivity. In this study, a high specific activity Cobetia marina ALP (CmAP) and an improved coupling method were used to develop an N-terminal pro-B-type natriuretic peptide (NT-proBNP) diagnostic reagent. (2) Methods: The purification method of CmAP was improved and the related enzyme activities were assessed. The enzyme and magnetic beads were coupled only to the Fc region of the detection antibody and the capture antibody, respectively, by using a specially improved method. The NT-proBNP in human serum was assessed. (3) Results: The specific activity of the purified CmAP was found to be 13,133 U/mg. No loss in the enzyme activity was observed after its storage at room temperature for 4 months. The sensitivity of the in vitro diagnostic reagents was found to be 0.58 ng/L. (4) Conclusions: CmAP can be applied as a substitute for the commercial ALP. Analytical parameters indicated that the chemiluminescence diagnostic reagent for NT-proBNP is adequately sensitive and reliable for detecting the serum NT-proBNP, which suggests that both the enzyme and coupling method are suitable for the CLEIA.