Catalytic Hairpin Assembly-Aided CRISPR/Cas-SERS for Nucleic Acid Detection Enhanced by Multiple Catalytic Amplification

Abstract

This study established a surface-enhanced Raman scattering (SERS) detection method based on the catalytic hairpin assembly (CHA) reaction combined with clustered regularly interspaced short palindromic repeats and its associated proteins (CRISPR/Cas), integrated with the guanine-quadruplexes/Hemin (G4/Hemin) catalytic amplification effect, which is available for identifying a typical indicator for genetically modified crops, the 35S promoter from the cauliflower mosaic virus (CaMV35S). Target CaMV35S-derived RNA (CaMV35S RNA) triggers the CHA reaction, yielding a double-stranded DNA product (C1/C2) that is recognized by CRISPR RNA (crRNA), thereby activating the trans-cleavage activity of Cas12a. A magnetic bead-based nanoprobe was fabricated by linking the hybridization chain reaction (HCR) product to the magnetic bead. This probe can fold into repeated G4 units under a specific potassium ion concentration to enable a high loading of Hemin, thereby providing strong peroxidase-like catalytic activity for 3,3′,5,5′-tetra­methyl­benz­idine (TMB). In a positive trial, the 35S RNA initiates CHA, and the CHA product activates Cas12a, disrupting the HCR products on the magnetic bead probe, preventing the G4 structure formation, accordingly yielding a weak SERS signal from TMB. In contrast, a negative trial fails to activate Cas12a, and the probe retains its nanoenzyme activity, resulting in a strong SERS signal from the produced oxTMB. Owing to the collateral trans-cleavage activity of Cas12a and the high peroxidase-like nanozyme activity of G4/Hemin, this method achieves the highly sensitive SERS detection of genetically modified samples without target gene preamplification.

Introduction

Due to the advantages of genetically modified (GM) crops, such as insect resistance and herbicide tolerance, their cultivation has expanded year by year, driving increased demand for testing. The core of nucleic acid identification in GM crops involves detecting foreign elements introduced during modification. The cauliflower mosaic virus 35S promoter (CaMV35S) is a widely used artificial promoter. Its core sequences have become important vectors for plant genetic engineering. Detection of the CaMV35S promoter allows determination of whether the product contains transgenic plant components. Thus, many nucleic acid analytical methods have been developed and applied to screen CaMV35S fragments to identify GM crops. −

CRISPR (clustered regularly interspaced short palindromic repeats), as a gene-editing technology, has significant potential for molecular diagnostics, especially in nucleic acid testing, due to its high sensitivity and specificity. Currently, the most common Cas proteins are Cas9, Cas12a, and Cas13a. Among these, Cas12a can be activated by dsDNA and cleaves single-stranded DNA (ssDNA) indiscriminately. − During actual sample testing, since extracted nucleic acids are in low content, preamplification of the target gene is usually needed. Methods such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), or recombinase polymerase amplification (RPA) are used to amplify the target sequence. However, amplification can cause some drawbacks, including longer testing time and a higher risk of false positives.

Both catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) are enzyme-independent, room-temperature amplification techniques with considerable potential for nucleic acid testing. The CHA reaction amplifies the signal by inducing the self-assembly of a DNA hairpin into a cyclic assembly of DNA hairpins into double-stranded products (dsDNA) under isothermal conditions, without the need for enzymes. The HCR reaction involves the alternating opening of DNA hairpin structures, initiated by the target molecule, under isothermal, enzyme-free conditions, forming linear dsDNA with numerous repeat units to achieve cascade signal amplification. During nucleic acid testing, very few target genes enable CHA and HCR reactions to produce dsDNA nanowires. These products generate signals in the CRISPR reaction after the target gene is recognized. − In a previous study, a CHA-CRISPR/Cas12a combined system was used to detect aflatoxin B1 (AFB1). It employed an AFB1 aptamer-driven CHA reaction, in which the reaction products activate the CRISPR/Cas12a system to perform trans-cleavage, thereby efficiently generating a fluorescent detection signal. The HCR-CRISPR/Cas12a combined system was also employed for detecting microRNA (miRNA). This method enabled miRNA-driven HCR nanowires to activate the CRISPR/Cas12a system, yielding highly sensitive fluorescent signal by trans-cleaving FAM-labeled probes. Existing amplification-assisted CRISPR sensing systems may rely either on enzyme-mediated target gene preamplification, such as PCR, RPA, and LAMP, or on enzyme-free nucleic-acid circuit amplification, such as CHA and HCR (Table S3). Usually, a multistep process is required to provide distinct solution environments and reaction conditions for target gene preamplification and CRISPR sensing.

To develop an integrated CHA-CRISPR/Cas12a recognition system, we established a surface-enhanced Raman spectroscopy (SERS) detection platform for GM crops by combining CHA-CRISPR/Cas12a with a multi-G4/Hemin nanoprobe, enabling the identification of CaMV35S RNA with high specificity and sensitivity. SERS is a unique optical phenomenon that achieves extremely high signal amplification, up to millions of times. It has been widely used for biosensing due to its high sensitivity, fingerprinting, and nondestructive detection capabilities, and is also applicable to aqueous-phase detection. When applied to the detection of GM corn, the combination of SERS with the CHA/HCR conjugation system enables target-preamplification-free detection of GM crops without PCR/RPA/LAMP-aided enzyme-mediated target gene preamplification. Several CRISPR-assisted SERS biosensors have recently been reported for nucleic acid detection, including CRISPR/Cas12a- or dCas9-mediated SERS strategies and CRISPR-assisted magnetic SERS/SERRS platforms (Table S4). These studies demonstrate the feasibility of combining CRISPR-based sequence recognition with Raman readout. However, most reported CRISPR-SERS assays still rely on enzyme-joined target-gene preamplification, such as PCR, RPA, or LAMP, or use CRISPR primarily as a recognition/transduction module coupled to conventional Raman reporters. In this work, the unique contribution is the integration of target-triggered CHA, Cas12a-mediated trans-cleavage, and an HCR-constructed multi-G4/Hemin magnetic nanoprobe into a single cascade for CaMV35S RNA analysis. The target RNA does not need to be replicated. Instead, it initiates CHA to generate the C1/C2 activator, which activates Cas12a/crRNA and subsequently modulates the nanoprobe’s catalytic activity, achieving signal-amplified colorimetric and SERS readouts. Our developed sensing strategy offers many advantages: (1) Dual-mode output. Both the SERS response and the chromogenic signal are obtained, enabling their use in various detection scenarios. (2) No target gene preamplification. Unlike methods that require PCR, RPA, or LAMP prior to CRISPR detection, the proposed assay directly analyzes the extracted target RNA without enzymatic amplification. The analytical approach is designed by introducing CHA, Cas12a trans-cleavage, and HCR-assisted G4/Hemin catalysis. (3) High pM-level sensitivity. This is mainly due to multiple signal-cascade amplifications, including CHA, a repeated G4/Hemin building block fabricated via an HCR reaction, and high Hemin loading to enable efficient TMB catalysis.

Experimental Section

Synthesis of Nanoprobes (Fe₃O₄-ssDNA and Fe₃O₄-H1/H2)

The nanoprobe we used in the present study is based on magnetic beads, which enable convenient washing and magnetic separation. First, magnetic beads with a carboxyl group surface (Fe₃O₄-COOH) were activated using the 1-ethyl-3-(3-(dimethyl­amino)­propyl)­carbo­diimide (EDC)/N-hydroxy­succin­imide (NHS) reaction. 200 μL of EDC (100 mM) and 200 μL of NHS (100 mM) were added to 200 μL of the Fe₃O₄-COOH solution (1.0 mg/mL). The mixture was incubated at 37 °C for 4 h. After the reaction, the magnetic beads were washed three times with water and resuspended in 500 μL of buffer. Subsequently, 10 μL of ssDNA (100 μM, the oligonucleotide sequence of ssDNA is provided in Supporting Information) was added to the system, and the mixture was incubated with shaking at room temperature for 4 h. Afterward, the magnetic beads were washed 3 times and resuspended in 100 μL of buffer. Thus, magnetic beads decorated with ssDNA were obtained. They were stored at 4 °C for further use.

The HCR reaction was initiated from the ssDNA on the magnetic beads. First, we incubated the hairpin 1 and 2 (H1, H2) sequences at 95 °C for 10 min, then at 4 °C for 20 min to form two hairpin structures. 5 μL of H1 and H2 (25 μM for each), and 10 μL of buffer (NaCl 100 mM, MgCl₂ 50 mM) were added to 5 μL of the above solution. The mixture was incubated at 37 °C for 2 h to perform the HCR reaction on the magnetic beads, producing HCR nanowires with repeated guanine (G) units. The magnetic beads were then washed three times with water to obtain the probe Fe₃O₄-H1/H2 with multiple G-rich branches. These G-rich sequences were folded into G-quadruplex (G4) structures by incubating Fe₃O₄-H1/H2 (10 μL) in 20 μL of a Tris-HCl buffer (pH 7.4, containing 80 mM KCl) at room temperature for 30 min. It should be noted that the K⁺ ion plays a key role in the formation of G4.

Verification of CHA and HCR Product Formation by PAGE

First, we incubated the C1 and C2 sequences at 95 °C for 10 min, then at 4 °C for 20 min to form two hairpin structures.

To initiate the CHA reaction, we added 1.5 μL of CaMV35S RNA, 3 μL of C1 (25 μM), and 3 μL of C2 (25 μM) to the PCR tube, and then the buffer was supplemented to a total volume of 25 μL. We incubated the mixture at 37 °C for 2 h. The products were then verified by polyacrylamide gel electrophoresis (PAGE) analysis.

The HCR reaction was completed as follows. We added 2 μL of ssDNA, 5 μL of H1 (25 μM), and 5 μL of H2 (25 μM) to a PCR tube, then supplemented the buffer to a total volume of 25 μL. After incubation of the mixture at 37 °C for 2 h, the products were verified by PAGE analysis.

Real-Time Fluorescence Validation of CHA-CRISPR Reactions

1.5 μL of CaMV35S RNA was added to a PCR tube, followed by 3 μL of C1 (25 μM), 3 μL of C2 (25 μM), 2 μL of buffer, 1 μL of Cas12a (0.5 μg/μL), 1 μL of crRNA (10 μM), 2 μL of MgCl₂ (40 mM), 2 μL of 10× PBS, 2 μL of fluorescent probe, and water to a final volume of 20 μL. The mixture was incubated at 37 °C for 60 min. FAM-ssDNA-TAMRA was selected as the fluorescent probe (F-Q reporter) at a concentration of 10 μM. Real-time fluorescence monitoring of Cas12a activation using an F-Q reporter on a quantitative PCR (qPCR) instrument­(Roche Life Sciences LightCycler® 96).

SERS Measurement

In this reaction system, different concentrations of 1 μL of CaMV35S RNA, 3 μL of C1 (25 μM), 3 μL of C2 (25 μM), 2 μL of buffer, 1 μL of Cas12a (0.5 μg/μL), 1 μL of crRNA (10 μM), 2 μL of MgCl₂ (40 mM), 2 μL of 10× PBS, and 10 μg of Fe₃O₄-H1/H2 probe were added. The mixture was then diluted to a final volume of 20 μL with ultrapure water. The mixture was incubated at 37 °C for 45 min.

After completing the CRISPR reaction, the magnetic beads were washed three times with ultrapure water. Then, 20 μL of Tris-HCl buffer (pH 7.4, 80 mM KCl) and 5 μL of Hemin (5 μM) were added to the system. The mixture was incubated in the dark at room temperature for 30 min. After the reaction, the microbeads were washed three times with a Tris-HCl buffer and then resuspended in 10 μL of a Tris-HCl buffer.

We added 10 μL of Fe₃O₄-G4/Hemin, 10 μL of (10 mM) H₂O₂, and 10 μL of (10 mM) TMB to the PCR tube and incubated it at room temperature for 15 min. Then, we mixed 1 μL of the reaction solution with 5 μL of silver nanoparticles (AgNPs, the procedures for preparing AgNPs are provided in Supporting Information) and conducted SERS testing. The integration time was 1 s, and the accumulation time was set to 1. In the specificity experiment, the CaMV35S RNA sequence was replaced with a mutated base sequence, while all other steps remained unchanged.

RNA Extraction from Corn Leaves

To measure the real samples, the cultivated corn leaves were collected with liquid nitrogen in a mortar. The CaMV35S RNAs from corn leaves were extracted using the SPARKeasy Plant RNA Kit (AC0305). First, we add 2 μL of the extracted RNA, 3 μL of C1 (25 μM), 3 μL of C2 (25 μM), 2 μL of buffer, 1 μL of Cas12a (0.5 μg/μL), 1 μL of crRNA (10 μM), 2 μL of MgCl₂ (40 mM), 2 μL of 10× PBS, and 10 μg of Fe₃O₄-H1/H2 probe to a PCR tube. Then, we added water to supplement the total volume to 20 μL. Next, the tube needs to be placed in a 37 °C incubator for 45 min. After completing the CRISPR reaction, the magnetic beads were washed three times with ultrapure water. Then, 20 μL of Tris-HCl buffer (pH 7.4, 80 mM KCl) and 5 μL of Hemin (5 μM) were added to the system. The mixture was incubated in the dark at room temperature for 30 min. After the reaction, the microbeads were washed three times with a Tris-HCl buffer and then resuspended in 10 μL of a Tris-HCl buffer. After that, qPCR was performed to confirm the genetic status of maize samples .

Results and Discussion

Principle of CHA-CRISPR-SERS Biosensors

Scheme illustrates a biosensor platform for detecting the transcription start site (TSS) of the CaMV35S promoter in transgenic maize and its downstream RNA. First, a magnetic bead-based nanoprobe was developed (Scheme b). The magnetic bead with the carboxyl group surface (Fe₃O₄-COOH, ∼200 nm) was activated with EDC/NHS, followed by the introduction of amino-modified ssDNA. The ssDNA in the Fe₃O₄-ssDNA probe can activate the HCR reaction, forming HCR nanowires on the magnetic bead. The resulting HCR nanowires form a chain with multiple repeated guanine (G)-rich nucleotide sequences that can assemble into a G4 in the presence of K⁺ (Scheme c). Hemin molecules can insert into these G4 units to form G4/Hemin complexes, which exhibit peroxidase-like nanoenzyme activity. Therefore, the G4/Hemin complex can oxidize TMB to oxTMB, accompanied by changes in SERS intensity and color (Scheme d). This nanoprobe functions as a substrate for Cas12a in this design. The repetitive G4/Hemin units on the nanoprobe can enhance catalytic efficiency and ensure high response sensitivity.

1. Principle of CHA-CRISPR/Cas12a-SERS Detection of GM Crops: (a) The CHA Cascade Reaction; (b) Synthesis of Magnetic Bead Nanoprobe; (c) Formation of Peroxidase-like Nanoenzyme; and (d) Sensing Mechanism .

^a The CaMV35S RNA activates the CHA cascade, enabling target gene recognition and yielding C1/C2 complex products.

^b HCR nanowires form repeated G4 in the presence of K⁺ ions, and G4 units can load Hemin with a high content.

^c CRISPR/Cas12a recognizes C1/C2 and induces Cas12a-dependent disruption of the G-rich Fe₃O₄-H1/H2 nanoprobe, thereby reducing G4/Hemin-mediated catalytic activity. Thus, it fails to catalyze the oxidation of TMB to oxTMB, resulting in a weak SERS signal from oxTMB. In the control or no-target RNA case, the G4/Hemin complex will remain, and its high peroxidase-like activity will catalyze TMB to oxTMB, resulting in a chromogenic reaction and a strong SERS signal when mixing the reaction solution with the Ag colloid.

The hairpin structures C1 and C2 are designed for the CaMV35S RNA. As shown in Scheme a, a single RNA chain binds to the sticky ends of C1, unfolding the C1 hairpin. Subsequently, C2 binds to the C1/RNA complex, displacing the RNA to form a C1/C2 complex. The displaced RNA initiates another reaction cycle, thereby sustaining cyclic CHA amplification. In short, only a very small number of target RNAs can trigger this CHA step, thereby enabling high detection sensitivity. Importantly, there is no preamplification of target RNA, thereby reducing the risk of target-amplification-related false-positive results that often occur in many amplification-aided detection methods. This C1/C2 complex serves as the recognition moiety for the CRISPR/Cas12a/crRNA system.

During GM detection, the presence of CaMV35S RNA triggers the CHA reaction. The resulting product activates Cas12a for cis-cleavage of the product and trans-cleavage of ssDNA within the system, causing Cas12a-dependent disruption of the Fe₃O₄-H1/H2 nanoprobe (Scheme d). This decreases TMB oxidation in the absence of peroxidase, resulting in no color change and a weak Raman signal from very few oxTMB products, even with the enhancement provided by the SERS-active silver colloid. Otherwise, Cas12a will remain silent in the target-gene-absent trials because the CHA reaction remains inactivated. As a result, the G4/Hemin on the magnetic beads maintains its geometry and catalytic activity, producing oxTMB, a chromogenic product that supports a strong SERS signal under silver nanoparticle enhancement.

This design differs from previously reported CRISPR-assisted SERS assays in that the CHA product is not used only as a hybridization product or reporter-binding scaffold, but serves as the activator for Cas12a/crRNA. Meanwhile, the Fe₃O₄-H1/H2 nanoprobe fabricated by HCR provides repeated G-rich units for G4/Hemin formation, enabling catalytic amplification of the oxTMB SERS signal. Therefore, the system integrates three functional processestarget-triggered CHA, CRISPR/Cas12a trans-cleavage, and G4/Hemin-mediated catalytic SERS readoutinto one cascade sensing system.

Characterization of the Magnetic Bead-Based Nanoprobe

The magnetic beads (Fe₃O₄-COOH, approximately 200 nm, Figure S1a) were modified with amino-modified ssDNA by the formation of amide bonds. Dynamic light scattering (DLS) results (Figure a) indicate that the surface charge of the magnetic beads shifted from −32.8 mV to −27.5 mV after EDC/NHS activation of the surface carboxyl groups. Upon binding to ssDNA, the surface charge shifted further to −35.7 mV, confirming the successful attachment of the ssDNA to the magnetic beads.

1.

Characterization of the nanoprobes. (a) Surface charge characterizations of Fe₃O₄, Fe₃O₄-EDC/NHS, and Fe₃O₄-ssDNA. The errors were obtained from the statistics of three parallel trials. (b) ssDNA-activated HCR reaction. Lane 1: ssDNA; Lane 2: H1; Lane 3: H2; and Lane 4: HCR product (ssDNA/H1/H2). The ssDNA:H1:H2 ratio in the HCR reaction was 2:5:5. (c) UV–vis absorption spectra of G4/Hemin formation in HCR nanowires in the presence of K⁺ and Hemin versus Hemin molecules. (d) SERS spectra of TMB and oxTMB under the enhancement of the Ag colloid with 633 nm laser irradiation (7.1 mW), an integration time of 1 s, and a single accumulation.

The ssDNA serves as the HCR primer, anchoring the H1 to the magnetic bead surface. The exposed toehold of H1 subsequently hybridizes with H2 and H1, triggering HCR and forming Fe₃O₄-H1/H2, with repetitive G-rich nucleotide sequences (Scheme b). To confirm the HCR product, we employed PAGE electrophoresis for ssDNA, H1, H2, and the HCR product in solution (Figure b). The molar ratio of ssDNA:H1:H2 was set as 2:5:5, and the reaction was performed at 37 °C for 2 h. Lane 4 shows distinct cascade bands corresponding to HCR products, in contrast to Lanes 2 (only H1) and 3 (only H2), confirming that the HCR reaction is feasible.

In the presence of K⁺, the complex H1/H2 on the magnetic bead will fold into G4 structures, into which Hemin molecules can insert. Upon Hemin binding to the G4, the UV–visible spectrum of Hemin exhibits a significant red shift from 407 to 418 nm (Figure c), confirming the G4/Hemin complex formation.

Figure d shows the SERS spectra of TMB and oxTMB. It is observed that oxTMB exhibits a strong signal at 1604 cm^–1. To perform the SERS measurement, we mixed 2 μL of the reaction solution with Ag colloid (approximately 70 nm, Figure S1b). The Raman peak assignments for oxTMB molecules are listed in Table S1. Figure S2 demonstrates the feasibility of the G4/Hemin complex catalyzing the TMB reaction by comparing the nanoprobe. Only the presence of G4/Hemin enables the catalyzed conversion of TMB to oxTMB. Because the amount of Fe₃O₄-H1/H2 nanoprobe affects the loading of HCR-constructed G-rich units and the subsequent G4/Hemin-mediated catalytic reaction, the nanoprobe dosage was optimized before analytical detection. As shown in Figure S3, the Fe₃O₄-H1/H2 amounts including 5, 10, and 20 μg were assessed, among which 10 μg of probes produced the strongest oxTMB SERS signal. Therefore, 10 μg of Fe₃O₄-H1/H2 nanoprobe was selected for subsequent CHA-CRISPR-SERS experiments.

Verification of CHA and Activation of Cas12a

The CaMV35S RNA triggers CHA without any enzymes, and the CHA product was confirmed by gel electrophoresis (12% native PAGE). In the CHA reaction, the CaMV35S RNA:C1:C2 ratio was set to 1:2:2, and the reaction was conducted at 37 °C for 2 h. Electrophoresis data are shown in Figure a. Lanes 1, 2, and 3 represent CaMV35S RNA, C1, and C2, respectively, while Lane 4 shows the CHA product C1/C2 complex. The upper band on the PAGE gel shows significant upward migration, whereas the bottom bands (C1 and C2) show marked fading. This phenomenon indicates that the designed sequences of C1 and C2 are valid to form the C1/C2 complex.

2.

Verification of CHA-mediated Cas12a activation and compatibility of the simultaneous co-incubation CHA-CRISPR reaction. (a) 12% PAGE analysis of target-induced CHA product formation. Lane 1: CaMV35S RNA; Lane 2: C1; Lane 3: C2; and Lane 4: CHA product generated by CaMV35S RNA-triggered C1/C2 assembly. (b) Schematic diagrams of sequential (left) and simultaneous CHA-CRISPR (right) procedures. In the simultaneous reaction, target RNA, C1, C2, Cas12a, and crRNA were mixed simultaneously. In the sequential reaction, CHA was first performed to generate the C1/C2 activator, followed by the addition of Cas12a/crRNA and F-Q reporter. (c) Real-time fluorescence comparison between simultaneous and sequential CHA-CRISPR reactions, recorded by qPCR. (d) End-point fluorescence intensity comparison between the simultaneous and the sequential CHA-CRISPR reactions. (e) Real-time fluorescence monitoring of Cas12a activation using a F-Q reporter on a qPCR instrument. The CHA-CRISPR group contains target RNA, C1, C2, Cas12a, and crRNA. The control groups includeC1-CRISPR, C2-CRISPR, and C1/C2-CRISPR, without target RNA.

To evaluate whether the coexistence of Cas12a/crRNA interferes with CHA intermediates, we compared simultaneous CHA-CRISPR co-incubation with a sequential CHA-CRISPR reaction (Figure b). In the simultaneous flow, target RNA (CaMV35S RNA), C1, C2, Cas12a, and crRNA were co-incubated during the same recognition reaction. In the sequential flow, CHA was first allowed to generate the C1/C2 activator, after which Cas12a/crRNA was added. Real-time fluorescence monitoring was performed using a real-time qPCR instrument and an F-Q reporter. As shown in Figure c, the simultaneous and sequential flows exhibited comparable fluorescence growth profiles and similar end-point fluorescence intensities (Figure d). These results indicate that the coexistence of Cas12a/crRNA with the CHA components did not markedly inhibit CHA progression or cause obvious premature disruption of CHA intermediates.

As stated above, the CHA chain product is the substrate for Cas12a. To validate recognition of CHA products (C1/C2 complex) by the crRNA-activated Cas12a, real-time fluorescence of the F-Q reporter was monitored. The CRISPR reactions were conducted separately to determine whether C1, C2, or a mixture of two could activate the trans-cleavage activity of Cas12a. As shown in Figure e, only the CHA-CRISPR reaction produces a fluorescent signal, indicating that the CHA products can activate trans-cleavage by Cas12a. Individual C1 or C2, as well as C1 plus C2 without the CaMV35S RNA, show no fluorescence. These data demonstrate that only the target RNA can initiate the CHA and that only the CHA product can activate the Cas12a/crRNA complex, thereby proving that our sensing strategy has high specificity for the target RNA.

Verification of CHA-CRISPR-SERS

To verify the contribution of each functional module, module-deletion controls were performed by omitting target RNA, C1, Cas12a, crRNA, or Hemin from the CHA-CRISPR-SERS system (Figure S4). In the absence of target-triggered CHA or an intact Cas12a/crRNA module, the Fe₃O₄-H1/H2 G4/Hemin retained its catalytic activity and generated strong SERS signals of oxTMB. In contrast, the complete target-containing system produced a markedly reduced SERS signal, supporting Cas12a-dependent regulation of the G4/Hemin catalytic module. These results confirm the cooperative roles of CHA, Cas12a, and the HCR-constructed G4/Hemin nanoprobe in the sensing cascade.

To further provide molecular evidence for Cas12a-mediated disruption of the HCR-constructed G4/Hemin nanoprobe, PAGE analysis was performed using the HCR-assembled H1/H2 structure as the cleavage substrate. As shown in Figure S5, H1, H2, the HCR product, and the CHA product show distinct electrophoretic bands. When the HCR product was incubated with the CHA product, crRNA, and Cas12a, the HCR-associated band was markedly weakened, while fragmented bands and phanic smearing appeared, indicating that the cleavage/disruption of the HCR-assembled structure by activated Cas12a occurred. In contrast, HCR incubated with Cas12a/crRNA in the absence of the CHA activator did not show obvious degradation, confirming that the disruption depended on CHA-mediated Cas12a activation. These PAGE results provide molecular evidence indicating that the activated Cas12a-mediated cleavage/disruption of the H1/H2 structure has begun, which accounts for the reduced catalytic activity of G4/Hemin.

Figure shows the SERS spectra of CaMV35S RNA at different concentrations (1 pM–10 nM), detected using our designed CHA-CRISPR-SERS method. As the RNA concentration gradually decreases, the SERS intensity of oxTMB at 1604 cm^–1 increases linearly. A linear relationship is observed in the concentration range from 10 nM to 1 pM. The SERS intensity at 1604 cm^–1 is represented by the equation y = −4940x + 27315. Thus, the limit of detection was calculated to be approximately 5.49 pM based on a signal-to-noise ratio of 3.

3.

Sensitivity testing of the detection method. (a) Validation of RNA activation of the CHA-CRISPR-SERS reaction at concentrations ranging from 10 nM to 1 pM. (b) A linear relationship between RNA concentration and triggered signal amplification. (c) Base mutation validation via SERS. (d) The specificity study for the CHA-CRISPR-SERS method using other RNA sequences instead of the target gene. Data are presented as mean ± standard deviation SD ( spectrum number n is 5). Statistical analysis is described in the Supporting Information.

To evaluate the sequence-discrimination capability of the assay, four artificially designed mismatch controls, RNA1–RNA4, containing one, two, three, and four nucleotide substitutions, respectively, were tested using our designed CHA-CRISPR-SERS method. The mutation positions were randomly selected within the CaMV35S target sequence and were not based on known natural polymorphisms or functional sites. Therefore, RNA1–RNA4 do not represent known naturally occurring plant or microbial sequences and were used only for a preliminary assessment of how the number of mismatches affects assay discrimination. In addition to mismatched RNA sequences, plant-derived and GM-related gene samples were included to further assess the assay’s specificity in the presence of GM plant-relevant backgrounds. As shown in Figures c and d, these non-target samples produce much stronger SERS intensities than the CaMV35S target RNA. The Raman intensities at 1604 cm^–1 are plotted, and it can be found that t clearly distinguish the target from non-target RNA samples can be clearly distinguished. These results further support the potential applicability of the assay for GM analysis in the presence of non-target plant-derived or GM-related nucleic acids.

Real Sample Analysis

Our sensing strategy was proof-of-concept tested using RNA extracted from fresh maize leaves. The maize samples were divided into three groups, GM1, GM2, and non-GM, provided by the Jilin Academy of Agricultural Sciences. Before CHA-CRISPR-SERS analysis, qPCR was used to authenticate the genetic status of the cultivated maize samples using genomic DNA as the template and CaMV35S-specific primers. As shown in Figure S6, GM1 and GM2 show earlier amplification signals than the non-GM samples, whereas the non-GM group shows only delayed fluorescence increases at later cycles. Next, qPCR was used only for qualitative sample authentication, not for quantitative sensitivity comparison with the proposed RNA-detection assay. After authentication, RNA was extracted from the maize leaves and analyzed using the CHA-CRISPR-SERS assay, as the procedures shown in Figure a. Each sample was measured five times, with 10 spectra collected per measurement. Figure b shows the SERS profiles of five randomly selected SERS spectra of three different samples. The SERS signal intensities at 1604 cm^–1 are plotted in Figure c, which shows significant differences. Figure d diplays the distinct color differences between the GM and non-GM systems due to oxTMB production. The SERS heatmaps for GM1, GM2, and non-GM trials (50 data points per group) are shown in Figure e, which present consistent oxTMB characteristic peaks and clear differences between GM and non-GM samples.

4.

Detection of actual transgenic corn samples. (a) Flowchart of the actual sample detection process. (b) Randomly selected SERS spectra of two transgenic corn varieties (GM1 and GM2) and non-GM. (c) Statistics of Raman intensity of oxTMB at 1604 cm^–1. Data are presented as mean ± standard deviation (n = 5 independent measurements). Statistical analysis was performed as described in the Supporting Information. (d) Chromogenic reaction results for GM maize (GM1, GM2), and non-GM control. (e) The heatmaps of SERS spectra for GM1, GM2, and non-GM control groups.

Conclusions

A CHA-CRISPR-SERS method integrating CHA-mediated generation of the Cas12a activator with CRISPR/Cas12a trans-cleavage was developed for RNA detection. A nanoprobe that enables multiple signal-cascade amplifications from a multi-G4/Hemin building block constructed via HCR was designed, and its highly efficient TMB-catalyzed activity yields high detection sensitivity at the pM level for RNA. The CHA-CRISPR/Cas12a recognition reaction is performed by simultaneous co-incubation at 37 °C, followed by magnetic separation and washing, K⁺/Hemin incubation, additional washing, TMB/H₂O₂ reaction, and silver nanoparticle-assisted SERS measurement. This method avoids PCR/RPA/LAMP-mediated, enzyme-mediated preamplification of the target gene, thereby reducing the risk of target-amplification-related cross-contamination. SERS and colorimetric readouts make this method convenient for various application scenarios, including lab tests and on-site assays, and it is promising for field detection when integrated with portable Raman instruments (see Figure S7). The high sensitivity of this assay is achieved by cascade signal amplification, including CHA-mediated generation of the Cas12a activator, Cas12a trans-cleavage, and HCR-assisted G4/Hemin catalytic amplification. These merits indicate that this approach may be extended to other nucleic acid targets after appropriate sequence design.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmeasuresciau.6c00142.

• Descriptions of materials and reagents, apparatus,preparation of Ag colloid, and statistical analysis; figures for TEM images of Fe₃O₄ and AgNPs, G4/Hemin peroxidase activity validation, nanoprobe dosage optimization, module-deletion and control experiments for the CHA-CRISPR-SERS system, Cas12a/crRNA trans-acting cleavage of HCR product, qPCR authentication of GM1, GM2, and non-GM maize samples, and detection method verification using a portable Raman spectrometer; and tables for oligonucleotide sequence and peak assignment of oxTMB (PDF)

L.L.: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. C.B.: Validation, Formal analysis, Data curation. H.W.: Validation, Formal analysis, Data curation. F.L.: Resources. Y.H.: Resources. X.L.: Resources, Funding acquisition. J.C.: Supervision, Funding acquisition. Y.Y.: Project administration, Resources. S.X.: Writing – review and editing, Supervision, Methodology, Funding acquisition, Conceptualization, Project administration.

The authors declare no competing financial interest.