CRISPR-Cas9 Activated Graphene Biointerfaces for Capture and Real-Time Monitoring of Cell-Free DNA on a Microneedle Patch

Recent advances in biointerfaces have led to the development of wearable devices that can provide insights into personal health. As wearable modules, microneedles (MNs) can extract analytes of interest from interstitial uid (ISF) in a minimally invasive fashion. However, some MNs are limited by their ability to perform high-effective extraction and real-time monitoring for macromolecule biomarkers simultaneously. Herein, we explored the synergetic effect of clustered regularly interspaced short palindromic repeats (CRISPR)-activated graphene biointerfaces, and reported an on-line CRISPR wearable microneedles patch for extraction and in vivo monitoring of Epstein-Barr virus cell-free DNA (EBV CfDNA) in ISF. This wearable system can orientally capture and directly quantify unamplied ISF CfDNA in vivo within 75 min, with anti-interference ability of 60%, and has good electrochemical stability within 3 days (RSD = 9.04%). The experimental results of immunodecient mouse models shows the feasibility and practicability of this proposed method. This wearable patch holds great promise for long-term in vivo monitoring ISF CfDNA and could be used for early disease screening and prognosis. The represented the timepoint. The skin stratum corneum of all BALB/c nude by disinfection with 75% ethanol, and smearing with 100 μL TE buffer (pH 8.0). The region of interest on mouse skin was dried with cotton. Finally, mice were plate the CRISPR MN wearable system as PCR (blood sampling by a commercial kit) in terms of qualitative analysis.


Introduction
With the arrival of the third medical revolution, wearable technology has experienced great development in the practice of modern personalized medicine. It is expected that wearable technology can become more streamlined, provide exibility, and be integrated into personal characteristics 1 . Wearable technology is a multidisciplinary subject that mainly includes soft materials, exible batteries, wireless signal communication, and chem-biosensing, which have garnered great attention for their ability to provide in-depth medical diagnostics and facilitate personal health assessments 2 . Although most efforts have focused on the analysis of small molecules or electrolytes [3][4][5][6][7] , the next generation of wearables aims for noninvasive or minimally invasive detection of macromolecular biomarkers (e.g., proteins or DNA) 2,4 . However, the greatest challenge for such wearables may be the effective extraction and realtime in vivo monitoring of these biomarkers.
Interstitial uid (ISF) is an ideal candidate for wearable chem-biosensors 4 . ISF contains a variety of biomarkers (e.g., protein, cells, nucleic acids) that are closely related to its blood concentration 2 . As an important component of wearable devices, microneedles (MNs) have been applied to ISF extraction in a safe, painless, and e cient manner 8- 10 . In our previous studies 11,12 , we proposed hydrogel MN patches to extract DNA from ISF and then analyzed them with portable exible electrochemical micro uidics. However, this approach was still an o ine monitoring method and would not be competent to the application of integrated wearable devices in real-world use. Therefore, the development of an online wearable system that can perform both sample extraction and real-time monitoring would be very important and could signi cantly improve personal health management.
Clustered regularly interspaced short palindromic repeats (CRISPR) technology has attracted extensive attention for the rapid analysis of nucleic acids speci cally and accurately due to its precise gene editing ability under the guidance of programmable single guide RNA (sgRNA) or CRISPR RNA (CrRNA) 13,14 .
Unlike zinc-nger nuclease and transcription activator-like effector nuclease technology, CRISPR uses CRISPR-associated (Cas) effectors to recognize and edit speci c gene sites, providing a promising method for sequence-speci c detection, such as HUDSON-SHERLOCK 15,16 and DETECTR 17  with a higher sensitivity of 0.16 fM 21 . Ampli cation-free CRISPR methodology could provide an effective and simple method for online nucleic acid wearable devices.
In this study, we propose CRISPR-Cas9 activated graphene biointerfaces on conductive MNs and combine them with reverse iontophoresis for the extraction and real-time monitoring of nucleic acids from ISF. Owing to the synergetic effect of CRISPR-Cas9 and graphene biointerfaces, the CRISPR-based wearable system was employed for real-time monitoring of Epstein-Barr virus cell-free DNA (EBV CfDNA), a biomarker released from nasopharyngeal carcinoma (NPC) 22,23 . This system has potential application value for real-time monitoring and early diagnosis of CfDNA-derived diseases.

Components, principle, and properties of the online CRISPR wearable patch
Here, we demonstrated an online CRISPR-Cas9 activated wearable patch based on the synergetic effect of CRISPR technology and graphene biointerfaces, where conductive MNs and reverse iontophoresis were employed for efficient extraction and realtime monitoring of EBV CfDNA from ISF in a minimally invasive fashion. A promising development in the study is the specific, continuous and direct monitoring of unamplified target DNA without preamplification (e.g., PCR or HCR). The CRISPR-activated wearable system includes the following modules: a flexible substrate, namely, a modified PDMS membrane; EBV CfDNA enrichment control, namely, a printed carbon nanotube (CNT)functionalized component; and real-time monitoring control, namely, a three-electrode prototype CRISPR-Cas9 MN system. Figure 1a, to achieve real-time monitoring of target DNA, the proposed wearable platform is composed of a spray-printed functional flexible patch and threeelectrode conductive MNs. First, the surface of the PDMS membrane was treated with plasma to increase the hydrophilicity of the membrane. Then, a hydrophilic membrane was Page 4/30 fabricated on the PDMS membrane via drop-casting of 1% chitosan solution. Due to the soft characteristics and weak surface adhesion of PDMS, the percolating microstructure would be deformed out of the interface during bending, stretching, and twisting 24 . Inspired by these properties, CNTs were deposited on the modified PDMS film by inkjet printing using a spray gun (0.17 MPa, 300 μm diameter) in this study 12 . The printed CNT pattern acted as a reverse iontophoresis compartment, separating negatively charged compounds (e.g., nucleic acids or ascorbate). Finally, a conductive CRISPR microneedle array as the working electrode was attached to the anode side of the CNT pattern. The CRISPR MNs showed three functions during real-time detection: (I) insertion into the epidermis to isolate and concentrate target DNA; (II) CRISPR gene editing specifically performed by Cas9/sgRNA immobilized on the surface of the CRISPR MNs; and (III) the formation of a three-electrode system to record electrical signals. Figure 1b shows a scheme of CRISPR MNs construction. In this CRISPR-Cas system, we used a catalytically inactivated Cas9 enzyme (dCas9) to form Cas9/sgRNA, denoted as dRNP 25 . Although both nuclease domains (RuvC and HNH) are deactivated in dCas9, the dRNP retain the ability to bind specifically to target DNA 13,26,27 . Immobilized dRNP can scan the entire DNA sequence under the guidance of sgRNA, where a 20-nt specific sequence matches the target DNA 14 . Once matched, dRNP can unwind the double-stranded helix and specifically bind with target DNA directly upstream of the 5'-NGG protospacer adjacent motif (PAM). The real-time monitoring capability of the wearable patch may come from two aspects: (I) dRNP of CRISPR-Cas9 as a driving force continuously searched and recognized target DNA; and (II) graphene biointerfaces on MNs provided highly efficient charged compound interactions and electron transport. In Figure 1c, hybridization of dRNP on the surface of graphene with CRISPR gene editing targets not only altered the conductivity of the graphene interface channel but also resulted in counterion accumulation. Therefore, an ion-permeable layer was generated on the graphene surface to maintain charge neutrality. The difference in ion concentration between the bulk solution and the ion-permeable layer produced the Donnan potential 28 .

As shown in
Hence, the recorded output electrical signals can reflect the real-time recognition of the  Figure S1).

Validation and affinity of dRNP to target DNA
To validate the feasibility of the CRISPR wearable system, we first tested the CRISPR-Cas9 reaction for EBV CfDNA gene editing in solution. From the genotyping data in Figure   2a-2b, two new bands in lane 1 were observed due to CRISPR gene editing, which contained Cas9, sgRNA and EBV CfDNA. Additionally, it was elucidated that the CRISPR reaction did not occur with mismatched sgRNA or sgRNA-free sequences. Accordingly, sgRNA plays an important role in the CRISPR-Cas system 14 . To this end, optimized experiments for sgRNA screening were performed in this study (Supplementary Information, Figure S2). The effect of the selected sgRNA on triggering CRISPR-Cas9 was verified in a concentrationindependent manner, as shown in Figure S2. According to region of interest (ROI) analysis of the PAGE gel results, the average ROI value of the CRISPR product bands gradually increased, while that of EBV CfDNA decreased ( Figure S2).
Then, we used a commercial solid microelectrode for EBV CfDNA target CRISPR gene editing on a skin chip (37 °C, pH 7.4). Figure 2c shows the original i-t curve data in response to 10 9 copies/μL EBV CfDNA. Compared with that of the control group, the fitting curve of EBV CfDNA was stable within 200 s and gradually increased after 400 s. The results showed that the current output signal comes from the directional recognition and binding of the target by the dRNP complex. In Figure 2d, there was a significant difference in the current between the positive and control groups, which was related to the appearance of the Donnan potential. These results might primarily demonstrate the proposed mechanism by which the dRNP compound immobilized on microneedles plays an important role in real-time online capture and monitoring of target DNA.
In this study, a CRISPR-Cas9 driving strategy was designed for wearable patches to monitor the CfDNA of ISF in real time. Therefore, the most important aspect is to ensure that dRNP has the ability to recognize and detect EBV CfDNA. For this purpose, we conducted experiments on a solid-state microelectrode (schematic in Figure S3). The targeting dRNP was modified on the surface of the microelectrode by a method similar to that used to prepare conductive microneedles. The CV and EIS characterization results To explore the quantitative analysis and real-time ability of this method, the CRISPR microelectrode was applied to test variable concentrations of EBV CfDNA.
According to reference 20 , we used Equation 1 as the unit of this real-time monitoring, where I response reflected the change between I t (measurement after incubation) and I b (calibration background before measurement).
In Figure 2h , where some EBV CfDNA on the interface was eluted, and the signal response value decreased. However, the NTC group did not show a corresponding signal response to these four processes. Similar to nucleic acid amplification (e.g., PCR) 29,30 , we hypothesized that there might be a defined time threshold for this protocol. The derivative of the real-time I response was obtained in Figure 2i, that is, dl/dt and CRISPR reaction time. The time threshold of this experiment was defined as ~12 min. That is, if there was no obvious change in the I response curve after ~12 min, it could be judged to be a negative sample.
To test whether this assay was quantitative, we defined a signal threshold for varying concentrations of EBV CfDNA. According to the derivative curve, we found that there was no significant change after 30 min, which was chosen as the signal limit. In Herein, dRNP immobilized on graphene biointerfaces could be used to trigger the event of target DNA detection without reagents or bulky equipment.
The above results primarily illustrated that dRNP on the surface of the microelectrode can recognize and bind target DNA. We were also interested in the binding constant between dRNP and EBV CfDNA; therefore, UV-vis spectrophotometry was employed to verify the interaction between the two 11 . As seen from the data in Figure   2k and 2l, the binding constant of K b =1.02 × 10 7 L/mol indicated that there was a good interaction between dRNP and EBV CfDNA. These results suggested that CRISPR-Cas9 can be employed in the subsequent microneedle array to achieve real-time monitoring.

Characterization and evaluation of the CRISPR wearable patch
In this study, we first fabricated conductive MNs using a series of simple and general methods, including drop casting and ion sputtering. The detailed preparation and optimization procedures are discussed in the Supplementary Information ( Figure S8). From the results of Figure S8, we found that the rigidity and modulus of the microneedles was closely relative to its shapes and inertial distance. In addition, to test whether the graphene biointerfaces on the MN surface were rigid enough to perform the CRISPR reaction, we compared the membranes under different conditions by scanning electron microscopy (SEM) (Supplementary Information, Figure S9). Figure 3a showed an off-the-shelf MNs that can be used directly for CRISPR-Cas9 decoration and wearable application.
As shown in Table S1, conductive MNs have been increasingly considered a promising tool for continuously monitoring from small molecules to biological macromolecules (e.g., RNA, DNA, protein), while it is still challenging to realize sample extraction and detection of nucleic acids simultaneously. In our research, reverse iontophoresis was used for preliminary enrichment and separation of the samples, which is an effective candidate for microneedles extraction function 12,31 . On this basis, real-time monitoring was performed by conductive MNs.
To test the quality of the prepared MNs, cyclic voltammetry (CV) was performed using [Fe(CN) 6 ] 3-/4as a probe, as shown in Figure 3b and 3c. From the data, it was observed that the area of the CV plot increased as the scanning rate increased. Two linear relationships between the scanning rate and redox peak current were obtained. The above results implied that the well-defined conductivity and mass transfer of the MNs was subject to a diffusion-limited mode 32 . Due to the high specific surface area, the prepared MNs To construct the wearable patch, poly dimethylsiloxane (PDMS, Sylgard 184, Dow Corning) was chosen as a candidate substrate due to its elastic and stretchable properties.
However, it is generally believed that the interface of PDMS is somewhat hydrophobic, which limits its application in wearable chem-biosensors 33 . One ideal method was to obtain the hydrophilic surface of PDMS by using stretchable and conductive nanomaterials, such as CNTs. Based on our previous report 12

In vitro extraction and real-time monitoring of EBV CfDNA using a CRISPR MN patch
The ultimate goal of the proposed real-time method was to realize proof-of-concept recognition of CfDNA on wearable MNs. It is essential to determine the anti-interference and sensitivity of this system. Thus, based on our previous report 12 , we used a simple skin chip to simulate human skin (37 °C, 10 V of reverse iontophoresis) as an in vitro real-time monitoring setup for the performance evaluation. As mentioned above, original conductive MNs were obtained for subsequent decorations, as shown in Figure 4a. Importantly, dCas9 was covalently immobilized, allowing the nuclease to bind tightly to the graphene surface.
The anti-interference of the CRISPR MNs was tested for the detection of 3×10 -12 M EBV CfDNA with different concentrations of fetal bovine serum (FBS) and control samples, including 0%, 10% and 60% FBS. The signal was recorded by i-t curve, as shown in Figure   4b. The CRISPR MNs generated a stable and well-defined current response with a relative standard deviation (RSD) of 2.49% under the interference of 10% FBS when compared to 0% FBS interference. Moreover, we observed that 60% FBS had an effect on the CRISPR MNs, and the RSD was 20.95%, but it still showed an "S" curve within 75 min. This capability could allow CRISPR MNs to be used for wearables in the real world.
We also investigated the real-time monitoring and sensitivity of the CRISPR MNs, as presented in Figure 4c. Under reverse iontophoresis on the skin chip, CRISPR MNs were applied for EBV CfDNA detection. In contrast to the NTC group, EBV CfDNA was recognized and bound by dRNP on the CRISPR MNs surface in the two positive groups, producing significant signal output. As the concentration of EBV CfDNA increased, the relative I response increased, which corresponded to the CRISPR microelectrode. From the result of i-t curves, we found that the signal tended to be stable within ~30 min, illustrating that the total monitoring time of 75 min is sufficient. The above results showed that the sensitivity was 3×10 -14 M.
As seen from the results in Figure    In earlier studies 23 , it was reported that NPC was asymptomatic at an early stage.
However, in numerous subsequent reports 22 , it was shown that NPC-related EBV CfDNA could be detected in NPC-positive patients. It has been proposed that EBV CfDNA was released by cell apoptosis and necrosis in patients with distant metastasis and localized diseases. Therefore, it is worthwhile to monitor circulating EBV CfDNA real time in an on-demand, minimally invasive and specific manner.
To avoid signal crossover, this CRISPR MN platform applied an intermittent measurement, similar to the GlucoWatch® biographer (Cygnus, Inc., Redwood City, CA, USA) 49 , as shown in Figure 5b. In brief, a voltage of 10 V was applied to extract the target for 3 min by reverse iontophoresis in the first step. Then, reverse iontophoresis was stopped, and the biosensor which remained to be laminated on the epidermis was engined for collecting electrochemical signal. The signal of this biosensor at the corresponding region was recorded for 1 min. These two steps were repeated to achieve real-time CfDNA monitoring.
As seen from the data in Figure  Unlike traditional labs, a wearable device is exposed to an uncontrolled environment for a long time, which might pose a challenge in detection accuracy during continuous monitoring 2 . Therefore, we conducted four independent tests on 18-day CNE-Luc-bearing BALB/c nude mice to verify the accuracy of the CRISPR MNs (Figure 5f, Figures S10-S11).
For the CRISPR MN platform, the procedure is shown in Figure 5b; for gold-standard PCR (kit provided by TIANGEN Co., Ltd., Beijing), the sampling blood was first treated by a commercial DNA extraction kit (provided by Sangon, Shanghai). Compared with PCR, CRISPR MNs ensured a reliable qualitative detection in mice, but their quantitative detection ability was not yet known.

Conclusion And Outlook
In summary, based on the synergetic effect of CRISPR-Cas9 and graphene biointerfaces, this study proposed an online wearable conductive MNs that performed high-effective extraction and real-time monitoring for NPC-derived CfDNA from ISF in vivo. This CRISPR-Cas9 activated wearable patch could continuously monitor cell-free DNA targets from ISF in vivo, with a detection limit of 1.1 fM (3δ b /K), with good electrochemical performance and stability within 3 days (RSD = 9.04%).
However, two major challenges still remain in terms of wearable devices: (I) due to the instability of the immobilized bioreceptor, the interface between the device and the active sensitive lm uctuates during the dynamic deformation process. Additionally, Joseph Wang et al. pointed out that the detection accuracy of wearables would be affected by the interface effect during continuous operation 2 . Unlike traditional laboratories, wearables are often exposed to harsh conditions that affect the bioactivity of immobilized receptors. Usage of a hydrogel or chitosan layer on the top to protect the immobilized bioreceptor may account for this issue. (II) Regarding sensitivity, probably due to the lack of preampli cation, the current version of an ampli cation-free strategy could not meet the requirements of highly sensitive DNA detection. As shown in Table 1, the optimized sensitivity proposed by Fozouni et al.
reached 0.16 fM for SARS-CoV-2 21 , and that of our proposed assay was 1.1 fM for target CfDNA, which was still unavailable for low-abundance biomarker analysis in particular practical applications (single copies/µL). Therefore, subsequent studies, including those on multiple Cas proteins, ordered mesoporous nanomaterials, precalibration processes, and metallic microneedle patches, should further endeavor to improve interfacial receptor immobilization and sensitivity to achieve detection of single-copy DNA of interest.

Synthesis of polymethyl vinyl ether-alt-maleic acid (PMVE/MA) hydrogel
The reagents were provided by Aladdin corporation (Shanghai). Brie y, rst, 10 g of PMVE/MA was dissolved in 60 mL of ddH 2 O and re ux-stirred for 24 hours at 80°C. After cooling, 6 g of polyethylene glycol (PEG, MW = 8000 Da) was added into the PMVE/MA solution for 12-hour re ux-stirring at 28°C.

Microneedles preparation
We designed the conceptual microneedle scheme, and a metallic wafer with a negative surface pattern was processed by Wuxi Guorui Electronic Technology Co., Ltd. (Wuxi, China). The metallic wafer was used to replicate the PDMS mould shape of the microneedle array (12×12 microneedles, microneedle base diameter 300 ± 10 µm, microneedle height 600 ± 50 µm, microneedle tip 30 ± 10 µm, microneedle interval distance 300 µm). To obtain the hydrogel-based microneedle patch, three-step replication processes were conducted in this study. First, the metallic wafer was ultrasonically cleaned with ddH 2 O for 3 min and then dried in an oven (80°C). Surface hydrophobic treatment was performed for metallic wafers for 5 min. PDMS was prepared at a weight ratio of base to curing agent of 10:1 and poured into the wafer. This metallic wafer mould was vacuumed (600 mmHg, 25°C, 30 min) to remove air from the PDMS matrix and centrifuged for 30 min (4000 rpm), which was repeated three times. Afterwards, this metallic wafer was placed in an oven (80°C, 4 h). The rst replication of the PDMS microneedle patch was carefully peeled from the metallic wafer. Next, Eco ex (smooth-on 0030) precursor mixture at a weight ratio of base to curing agent of 1:1 was poured carefully onto the PDMS master after surface hydrophobic treatment, which was vacuumed for 5 min to eliminate bubbles and cured at 80°C for 4 h. Thus, the second replication was achieved. Finally, the synthesized PMVE/MA hydrogel (13 mL) in this study was poured into a holder where the Eco ex master was xed on the bottom. This holder was rstly placed in a vacuum oven (80°C) for 7 h to ensure that the mixture solution lled the microwells. Then, the holder was transferred to an oven (90°C, 24 h). Consequently, the holder was placed in a fume hood and peeled off immediately. A pristine hydrogel microneedle patch was obtained and stored in a desiccator at 20°C when not in use.
For conductive MNs, the pristine MNs was rstly plasma-treated for 1 min. And 100 µL 0.5 mg/mL Cr dispersion solution in 0.1% chitosan was drop-casted on the surface of MNs immediately. And it was placed in oven (60°C, 30 min) to dry. Afterwards, a compact gold lm was formed on its surface by Au spurring (10 mA, 600s). Then, a gold wire (diameter of 200 µm) was attached to the contact area of the MNs by brushing carbon paste (SPI Supplies Co., USA) and placed in the oven (60°C, 10 min). After, it was coated a gold lm by Au spurring to maintain a consistent surface. Insulation and package step were applied for the contact and non-conductive area by Eco ex (smooth-on 0030) precursor mixture at a weight ratio of base to curing agent of 1:1. Finally, it was placed in oven (60°C, 60 min). A conductive MNs was obtained. For reference MNs, the conductive MNs was pasted with Ag/AgCl ink (BAS Inc., Japan); for counter MNs, the conductive MNs was pasted with carbon paste (SPI Supplies Co., USA). Thus, a three-electrode MNs system was ready for subsequence modi cation and application. The morphology and thickness of the MNs was validated by a stylus pro ler (AlphaStep D-600, KLA-Tencor Corp.).

Iontophoretic wearable patch construction
Firstly, the crude PDMS precursor mixture at a weight of base to curing agent of 6g:0.6g was poured carefully onto the square plain petri dish master (diameter of 9 cm, Corning corporation) after surface hydrophobic treatment, which was vacuumed for 5 min to eliminate bubbles and cured at 80°C for 1h.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. MovieS1.mp4 MovieS2.mp4 SupplementaryInformation.docx