Professor University of Connecticut Health Center, United States
Introduction:: Liver cancer is a global health concern, ranking as the fifth most common malignancy and the third leading cause of cancer-related deaths.[1] Alpha-fetoprotein (AFP) is a crucial protein biomarker used for liver cancer assessment.[2] Traditional methods of AFP detection, such as enzyme-linked immunosorbent assays (ELISAs), require sophisticated equipment and trained personnel, limiting their use for rapid screening in the population. The CRISPR-Cas biosensing system, known for its simplicity, sensitivity, and specificity, has gained attention for molecular diagnostics in various diseases.[3]
Here, we present a simple, affordable, and portable CRISPR-powered biosensing platform using a personal glucose meter (PGM) for quantitative detection of AFP in serum samples. The biosensor combines the high affinity of aptamers with the collateral cleavage activity of CRISPR-Cas12a, enabling sensitive and specific detection of AFP. To eliminate the need for expensive optical detection equipment, we integrated invertase-catalyzed glucose production with the glucose biosensing technology, similar to home-based diabetes testing, for quantifying AFP biomarker. We further validated the clinical utility of the CRISPR-powered personal glucose meter biosensor by determining AFP levels in clinical serum samples from liver cancer patients. The accuracy, portability, and cost-effectiveness of this biosensor offer a promising approach for point-of-care screening and detection of AFP and potentially other tumor biomarkers.
Materials and Methods:: Preparation of functionalized MBs
The mixture of aptamer and DNA activator in nuclease-free water was heated and then gradually cooled down to obtain the APT273–activator duplex. Streptavidin MBs were redispersed in Tris-HCl buffer containing APT273–activator duplex and then stirred to prepare APT273–activator duplex-modified MBs. Single-stranded DNA (ssDNA) linker was activated by TCEP and invertase was activated by SMCC. Activated invertase and ssDNA linker were stirred at room temperature to prepare biotin-ssDNA-invertase. Purified streptavidin MB was delivered into biotin-ssDNA-invertase and continuously stirred to prepare invertase-modified MBs.
Detection of AFP biomarker in spiked serum and clinical samples
5% human serum was incubated with MB-aptamer-activator suspension. Following magnetic separation, supernatant was transferred into a new tube to prepare CRISPR-Cas12a reaction system, which contained LbCas12a, crRNA, 1× NEBuffer 2.1and MB-ssDNA-invertase. After magnetic separation, supernatant in the CRISPR-Cas12a reaction tube was transferred into another tube to prepare the glucose-producing reaction system containing sucrose buffer. After incubation at 37°C, the glucose concentration of the mixed solution was measured by a PGM.
Results, Conclusions, and Discussions:: Results and Discussions
Design and Construction of the CRISPR-powered PGM Biosensor for AFP Detection
The workflow of AFP detection using a CRISPR-powered PGM biosensing platform is illustrated in Figure 1. This platform involves three main steps: A) AFP-induced activator release and magnetic separation from MB-aptamer-activator, B) CRISPR-Cas12a activation by the released activator and cleavage of MB-ssDNA-invertase, and C) glucose generation from sucrose hydrolysis caused by the released invertase and quantitative detection by the PGM.
Analytical performance and clinical validation of the CRISPR-powered PGM biosensor
Serial dilution of AFP biomarker ranging from 100 μg/mL to 0.01 μg/mL in human serum solution was tested. The PGM biosensor cannot detect 0.01 μg/mL AFP biomarker with an incubation time of 30 min because the generated glucose concentration was below the limit of detection of the PGM (20 mg/dL) (Figure 2). After a 60-min incubation, the biosensor could consistently detect 0.01 μg/mL (or 10 ng/mL) of AFP biomarker in serum samples. The relationship between glucose readouts and AFP concentrations was revealed through logarithmic fitting curves (Figure 3). The clinical application of CRISPR-powered PGM biosensor to detect AFP biomarkers was further investigated using eight samples from liver cancer patients and three samples from healthy individuals. The CRISPR-powered PGM biosensor did not detect glucose signal from serum of healthy controls while all eight patients’ samples exhibited significantly high glucose signals. AFP quantified in each patient sample using the established fitting curve was comparable to the conventional ELISA method (Figure 4).
Conclusions
The study introduces a novel biosensing platform that uses CRISPR-Cas12a, aptamer-based molecular recognition, and glucose biosensing to detect AFP biomarkers in human serum samples. With a wide detection range of 100 μg/mL to 0.01 μg/mL and a detection sensitivity of 0.01 μg/mL, the platform demonstrates high sensitivity and accuracy in quantifying AFP levels. The clinical utility of the platform is validated through testing of clinical samples, showing comparable performance to the conventional ELISA system. The simplicity and low cost of the biosensing platform make it a promising candidate for point-of-care applications, with potential implications for diagnosis and monitoring of diseases such as liver cancer.
Acknowledgements (Optional): : The work was supported, in part, by NIH R01EB023607 and startup funds at the University of Connecticut Health Center.
2. F. Trevisani, F. Garuti, A. Neri, Alpha-fetoprotein for diagnosis, prognosis, and transplant selection, Seminars in Liver Disease, Thieme Medical Publishers2019, pp. 163-77.
3. J.S. Gootenberg, O.O. Abudayyeh, M.J. Kellner, J. Joung, J.J. Collins, F. Zhang, Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6, Science, 360(2018) 439-44
