Wearable technology is rapidly advancing personalized medicine, aiming for non-invasive or minimally invasive detection of macromolecular biomarkers like proteins and DNA. Interstitial fluid (ISF), readily accessible via microneedles (MNs), contains various biomarkers. While previous studies utilized hydrogel MN patches for DNA extraction, offline monitoring limited their application in integrated wearable devices. Real-time monitoring is crucial for clinical applications, offering continuous health insights and improved self-management. CRISPR technology, with its precise gene-editing capabilities, provides high specificity and accuracy for nucleic acid analysis. Amplification-free CRISPR strategies further simplify the process for online wearable devices. This study aimed to develop an online wearable system capable of both sample extraction and real-time monitoring of cfDNA using CRISPR-Cas9 technology and graphene biointerfaces.

Existing wearable technologies predominantly focus on small molecules and electrolytes. The challenge lies in effectively extracting and monitoring macromolecular biomarkers in vivo. Microneedles offer a minimally invasive solution for ISF extraction. Previous research utilized hydrogel MN patches for DNA extraction and subsequent offline analysis. However, real-time in vivo monitoring of macromolecules remains a significant hurdle. CRISPR technology, particularly amplification-free methods, presents a promising avenue for sensitive and specific nucleic acid detection in wearable biosensors. Several studies have demonstrated the application of CRISPR-Cas systems in amplification-free detection of nucleic acids, showcasing their potential for sensitive and specific detection even at low concentrations.

This study developed an online CRISPR-Cas9 activated wearable patch. The system comprised a flexible PDMS membrane modified for hydrophilicity using plasma treatment and chitosan, a printed carbon nanotube (CNT)-functionalized component for cfDNA enrichment via reverse iontophoresis, and a three-electrode CRISPR-Cas9 MN system for real-time monitoring. CRISPR MNs were fabricated using a series of methods, including metallization and CRISPR system functionalization. The dCas9 enzyme, lacking nuclease activity but retaining target DNA binding ability, was immobilized on the graphene surface of the MNs along with a sequence-specific sgRNA. The binding of the dRNP complex to target cfDNA modified the graphene interface conductivity, generating a measurable electrical signal. The system's performance was validated in vitro using a commercial solid microelectrode and a skin chip mimicking human skin. The CRISPR MN patch's mechanical properties were characterized using various tests including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and mechanical stress-strain analysis. In vivo studies were conducted using an EBV-mice model to assess the patch's ability to monitor cfDNA in real-time. Finally, the system's versatility was demonstrated by applying it to the detection of cfDNA associated with sepsis and kidney transplantation.

The CRISPR-Cas9 activated wearable patch demonstrated high specificity and sensitivity in detecting cfDNA in vitro and in vivo. In vitro experiments using a commercial solid microelectrode showed a significant current difference between the positive and control groups, validating the proposed mechanism. The system exhibited a linear relationship between the current response and EBV cfDNA concentration in the range of 30–30,000 fM, with a detection limit of 1.1 fM. The CRISPR MNs showed excellent electrochemical performance and stability, with an RSD of 9.04% over 3 days. The wearable patch demonstrated stable electrical performance under mechanical distortions such as stretching, twisting, and bending. Histological analysis of piglet skin confirmed the microneedles' ability to penetrate the epidermis. In vivo experiments using an EBV-mice model demonstrated that the system could successfully monitor EBV cfDNA in real-time for up to 10 days, showing a strong correlation with bioluminescence imaging results. The system also successfully monitored sepsis-associated and kidney transplantation-associated cfDNA, highlighting its versatility. The patch showed good anti-interference capabilities, maintaining a stable current response even in the presence of 60% fetal bovine serum.

This study successfully developed a novel wearable microneedle patch for real-time in vivo monitoring of cfDNA, addressing the limitations of existing offline methods. The integration of CRISPR-Cas9 technology and graphene biointerfaces enabled high specificity and sensitivity, overcoming challenges associated with detecting low-abundance biomarkers in complex biological matrices. The real-time monitoring capability of this system offers significant advantages over traditional methods, providing continuous and dynamic information about disease progression. The ability to monitor multiple cfDNA targets highlights its potential for broad applications in various disease diagnostics. The in vivo results in immunodeficient mice demonstrate the feasibility and practicality of this method for long-term monitoring.

This study successfully demonstrated a programmable CRISPR-Cas9 microneedle patch for the long-term capture and real-time monitoring of universal cell-free DNA. The system showed high sensitivity and specificity, with good stability over 10 days in vivo. Future work will focus on further improving sensitivity and exploring its clinical applications in human subjects.

While this study demonstrated the effectiveness of the CRISPR MN patch in immunodeficient mouse models, further studies are needed to validate its performance in diverse physiological conditions. The study used a simulated ISF environment, which may differ from the actual composition of human ISF. Additionally, the long-term stability was demonstrated in vitro and in vivo within limited durations; extended in vivo studies are required to confirm the long-term stability and biocompatibility of the device in humans. Finally, the quantitative detection capability of the CRISPR MNs needs further improvement for more precise clinical applications.