A Highly Sensitive CRISPR‐Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar‐Level Real‐Time Quantification

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR) molecular system has emerged as a promising technology for the detection of nucleic acids. Herein, the development of a surface plasmon resonance (SPR) sensor that is functionalized with a layer of locally grown graphdiyne film, achieving excellent sensing performance when coupled with catalytically deactivated CRISPR‐associated protein 9 (dCas9), is reported. dCas9 protein is immobilized on the sensor surface and complexed with a specific single‐guide RNA, enabling the amplification‐free detection of target sequences within genomic DNA. The sensor, termed CRISPR‐SPR‐Chip, is used to successfully analyze recombinant plasmids with only three‐base mutations with a limit of detection as low as 1.3 fM. Real‐time monitoring CRISPR‐SPR‐Chip is used to analyze clinical samples of patients with Duchenne muscular dystrophy with two exon deletions, which are detected without any pre‐amplification step, yielding significantly positive results within 5 min. The ability of this novel CRISPR‐empowered SPR (CRISPR‐eSPR) sensing platform to rapidly, precisely, sensitively, and specifically detect a target gene sequence provides a new on‐chip optic approach for clinical gene analysis.

and then added dropwise into the above-mixed solution for 6 h. The reaction system was maintained at 60°C for 48 h. Upon completing the acetylenic coupling reaction, homogenous graphdiyne films formed on the surface of SPR chips, which were washed with acetone and ethanol, dried with N 2 , and stored in the dark until further use. The graphdiyne flakes suspended in the reaction solution were also collected, washed, and vacuum-dried for further characterization.

Site-directed mutagenesis
First, 1.5 μL of primers (10 μM), 1 μL of plasmid DNA (20 ng/μL), 13.4 μL of the reaction mix, and 34.1 μL nuclease-free water were mixed well and incubated at 37°C for 12 min to complete the DNA methylation reaction. Mutagenesis PCR was then performed with pre-denaturation (94°C for 2 min); 18 thermal cycles of denaturation at 94°C for 20 s, annealing at 57°C for 30 s, and extension at 68°C for 1 min; and final extension at 68°C for 5 min. To enhance the mutagenesis efficiency, 4 μL of the resulted product was incubated with 16 μL of the enzyme mix at room temperature for 10 min to remove unmethylated templates. The reaction was terminated with the addition of a 1 µL ethylenediaminetetraacetic acid solution (0.5 M), and the recombinant plasmid DNA was obtained for the transformation process.
In a typical transformation procedure, a vial of 50 µL DH5-T1 competent cells (Thermo Fisher Scientific, Waltham, MA, USA) was thawed on ice within 20 min and 2 µL of the recombinant plasmid DNA was gently pipetted into the vial. The mixture was incubated on ice for 12 min, followed by incubating in a water bath for exactly 30 s at 42°C. Each vial of cells was cooled down on ice for 2 min and incubated with 250 µL of pre-warmed SOC medium (Thermo Fisher Scientific) at 37°C for 1 h in a shaking incubator (225 rpm). The transformed cells were diluted by 20 times with SOC medium and 100 µL of the diluted cell suspension was inoculated to a Luria-Bertani-ampicillin plate using a steel-made scraper. The inoculated plates were inverted and incubated at 37°C for 16-20 h. Three colonies on each plate were picked up for further fermentation and plasmid isolation.

Material characterization
Low-resolution transmission electronic microscopy and high-resolution transmission electron microscopy images were captured by a JEM 2100 (JEOL, Japan) and FEI Tecnai TF30 (Thermo Fisher Scientific, Waltham, MA, USA) microscope, respectively. Nanoscale morphology was observed using atomic force microscopy (Bruker Dimension Ico). The X-ray photoelectron spectroscopy data were collected using an ESCALAB 250Xi XPS spectrometer (Thermo Fisher Scientific). Polarized Raman spectroscopy was carried out using an iHR 320 spectrometer (Horibai, Japan).
The concentration of all DNA samples was determined with Nanodrop UV-Vis spectrophotometers (Thermo Fisher Scientific). The concentration of all protein samples was measured using a BCA assay kit (Thermo Fisher Scientific) with an Epoch microplate reader (BioTek, USA). Thermal cycling was carried out for 2 min at 98°C for initial denaturation, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 68°C for 30 s. The melting curve was recorded from 60°C to 95°C. As a negative control, PCR amplicons were incubated with the empty dCas9-functionalized MBs (without sgRNA), and the cycle threshold (Ct) value of the supernatant was used as the quantification reference.

Magnetic bead separation, electrophoresis, and qPCR analysis
Furthermore, capture efficiency was calculated by the Ct value according to the following equation (1): where Ct sample is the Ct value of the target DNA sample that remained in the supernatant after incubating with CRISPR-MBs (colored in amplification curves), and Ct reference is the Ct value obtained from the negative control (black in amplification curves).
The DNA fragments with different target sequences were prepared by PCR with the pUC19 plasmid mutagens (Origin, Mut1, Mut2, and Mut3) and pCDH vector (GFP-Pos1, GFP-Pos2, and GFP-Pos3). Primers for each target sequence were designed by Primer Premier 6.0 (Tables S5-S8 (depending on the product length).

Verification of the immobilization of dCas9 and sgRNA
The chip-immobilized dcas9 protein was quantified using a modified method based on a similar principle with ELISA. After the immobilization using different concentrations of dCas9 protein solution on the graphdiyne chips, the chips were rinsed and trimmed into a proper size and put into a 96-well plate. After blocking with Blocking One buffer (Nacalai Tesque, Inc.) for 2 h, the chips were incubated with a rabbit monoclonal anti-CRISPR-Cas9 antibody (Abcam, ab189380) for overnight at 4°C, followed by rinsing and incubating with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG H&L secondary antibody (Abcam, ab6721) for 2 h at room temperature. Rinsed, and incubated with a luminol-based enhanced chemiluminescent substrate (SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermofisher Scientific, 34095). Finally, the absorbance at 450 nm of each well was read by using a microplate spectrophotometer (Epoch).
The anchored sgRNA was quantified following a enzymatic release using proteinase K. After the immobilization of dCas9 and different concentrations of sgRNA, the chips were rinsed and incubated with 1 μL proteinase K in 20 μL rinsing buffer at 60 ℃ 8 under gently shaking for 10 min. Also, a chip incubated without proteinase K was used for comparison, and all the buffer was collected for the following analysis.
Agarose gel electrophoresis was performed with 2.5% agarose gel in TAE buffer (pH

Binding kinetics parameters
To investigate the difference of binding kinetics among the varied target sites, the concentration of the pUC19 and pCDH amplicons was maintained at 1000 ng/μL. The molecular kinetic interactions can be described as the following equation: where k a is the association rate constant; k d is the dissociation rate; and C p , C A , and C PA represent the concentration of the probe, analyte, and their molecular complex, respectively. By introducing the relative SPR response change R, equation (2) can be rewritten as: where the maximum SPR response R max can be inferred by the results for the DNA analyte at 1000 ng/μL when CRISPR-SPR-Chip reached saturation. The equilibrium constant K D is determined using equation (4): = / (4).        Table S7. Primers for PCR amplification of pCDH-2k and pCDH-5k. Different amplification regions were selected to obtain the amplicons with and without the GFP gene.