Targeting MFGE8 secreted by cancer-associated fibroblasts blocks angiogenesis and metastasis in esophageal squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) play vital roles in establishing a suitable tumor microenvironment. In this study, RNA sequencing data revealed that CAFs could promote cell proliferation, angiogenesis, and ECM reconstitution by binding to integrin families and activating PI3K/AKT pathways in esophageal squamous cell carcinoma (ESCC). The secretions of CAFs play an important role in regulating these biological activities. Among these secretions, we found that MFGE8 is specifically secreted by CAFs in ESCC. Additionally, the secreted MFGE8 protein is essential in CAF-regulated vascularization, tumor proliferation, drug resistance, and metastasis. By binding to Integrin αVß3/αVß5 receptors, MFGE8 promotes tumor progression by activating both the PI3K/AKT and ERK/AKT pathways. Interestingly, the biological function of MFGE8 secreted by CAFs fully demonstrated the major role of CAFs in ESCC and its mode of mechanism, showing that MFGE8 could be a driver factor of CAFs in remodeling the tumor environment. In vivo treatment targeting CAFs-secreting MFGE8 or its receptor produced significant inhibitory effects on ESCC growth and metastasis, which provides an approach for the treatment of ESCC.

Enhanced mitophagy driven by ADAR1-GLI1 editing supports the self-renewal of cancer stem cells in HCC.

BACKGROUND AND AIMS: Deregulation of adenosine-to-inosine editing by adenosine deaminase acting on RNA 1 (ADAR1) leads to tumor-specific transcriptome diversity with prognostic values for HCC. However, ADAR1 editase-dependent mechanisms governing liver cancer stem cell (LCSC) generation and maintenance have remained elusive. APPROACH AND RESULTS: RNA-seq profiling identified ADAR1-responsive recoding editing events in HCC and showed editing frequency of GLI1 , rather than transcript abundance was clinically relevant. Functional differences in LCSC self-renewal and tumor aggressiveness between wild-type (GLI1 wt ) and edited GLI1 (GLI1 edit ) were elucidated. We showed that overediting of GLI1 induced an arginine-to-glycine (R701G) substitution, augmenting tumor-initiating potential and exhibiting a more aggressive phenotype. GLI1 R701G harbored weak affinity to SUFU, which in turn, promoted its cytoplasmic-to-nuclear translocation to support LCSC self-renewal by increased pluripotency gene expression. Moreover, editing predisposed to stabilize GLI1 by abrogating ß-TrCP-GLI1 interaction. Integrative analysis of single-cell transcriptome further revealed hyperactivated mitophagy in ADAR1-enriched LCSCs. GLI1 editing promoted a metabolic switch to oxidative phosphorylation to control stress and stem-like state through PINK1-Parkin-mediated mitophagy in HCC, thereby conferring exclusive metastatic and sorafenib-resistant capacities. CONCLUSIONS: Our findings demonstrate a novel role of ADAR1 as an active regulator for LCSCs properties through editing GLI1 in the highly heterogeneous HCC.

LOXL1 promotes tumor cell malignancy and restricts CD8 + T cell infiltration in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer mortality globally. Lymph node metastasis and immunosuppression are main factors of poor prognosis in CRC patients. Lysyl oxidase like 1 (LOXL1), part of the lysyl oxidase (LOX) family, plays a yet unclear role in CRC. This study aimed to identify effective biomarkers predictive of prognosis and efficacy of immunotherapy in CRC patients, and to elucidate the prognostic value, clinical relevance, functional and molecular features, and immunotherapy predictive role of LOXL1 in CRC and pan-cancer. METHODS: Weighted gene co-expression network analysis (WGCNA) was employed to explore gene modules related to tumor metastasis and CD8 + T cell infiltration. LOXL1 emerged as a hub gene through differential gene expression and survival analysis. The molecular signatures, functional roles, and immunological characteristics affected by LOXL1 were analyzed in multiple CRC cohorts, cell lines and clinical specimens. Additionally, LOXL1's potential as an immunotherapy response indicator was assessed, along with its role in pan-cancer. RESULTS: Turquoise module in WGCNA analysis was identified as the hub module associated with lymph node metastasis and CD8 + T cell infiltration. Aberrant elevated LOXL1 expression was observed in CRC and correlated with poorer differentiation status and prognosis. Molecular and immunological characterization found that LOXL1 might mediate epithelial-mesenchymal transition (EMT) process and immunosuppressive phenotypes of CRC. Functional study found that LOXL1 enhanced tumor cell proliferation, migration and invasion. Moreover, high LOXL1 levels corresponded to reduced CD8 + T cell infiltration and predicted poor clinical outcomes of immunotherapy. Similar trends were also observed at the pan-cancer level. CONCLUSIONS: Our findings underscore the critical role of LOXL1 in modulating both malignancy and immunosuppression in CRC. This positions LOXL1 as a promising biomarker for predicting prognosis and the response to immunotherapy in CRC patients.

The Development of Aptamer-Based Gold Nanoparticle Lateral Flow Test Strips for the Detection of SARS-CoV-2 S Proteins on the Surface of Cold-Chain Food Packaging.

The COVID-19 pandemic over recent years has shown a great need for the rapid, low-cost, and on-site detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, an aptamer-based colloidal gold nanoparticle lateral flow test strip was well developed to realize the visual detection of wild-type SARS-CoV-2 spike proteins (SPs) and multiple variants. Under the optimal reaction conditions, a low detection limit of SARS-CoV-2 S proteins of 0.68 nM was acquired, and the actual detection recovery was 83.3% to 108.8% for real-world samples. This suggests a potential tool for the prompt detection of SARS-CoV-2 with good sensitivity and accuracy, and a new method for the development of alternative antibody test strips for the detection of other viral targets.

Aptamers dimerization inspired biomimetic clamp assay towards impedimetric SARS-CoV-2 antigen detection.

Antigen-detecting rapid diagnostic testing (Ag-RDT) has contributed to containing the spread of SARS-CoV-2 variants of concern (VOCs). In this study, we proposed a biomimetic clamp assay for impedimetric SARS-CoV-2 nucleocapsid protein (Np) detection. The DNA biomimetic clamp (DNA-BC) is formed by a pair of Np aptamers connected via a T20 spacer. The 5'- terminal of the DNA-BC is phosphate-modified and then anchored on the surface of the screen-printed gold electrode, which has been pre-coated with Au@UiO-66-NH2. The integrated DNA-material sensing biochip is fabricated through the strong Zr-O-P bonds to form a clamp-type impedimetric aptasensor. It is demonstrated that the aptasensor could achieve Np detection in one step within 11 min and shows pronounced sensitivity with a detection limit of 0.31 pg mL-1. Above all, the aptasensor displays great specificity and stability under physiological conditions as well as various water environments. It is a potentially promising strategy to exploit reliable Ag-RDT products to confront the ongoing epidemic.

Cancer Stem Cells in Hepatocellular Carcinoma: Intrinsic and Extrinsic Molecular Mechanisms in Stemness Regulation.

Hepatocellular carcinoma (HCC) remains the most predominant type of liver cancer with an extremely poor prognosis due to its late diagnosis and high recurrence rate. One of the culprits for HCC recurrence and metastasis is the existence of cancer stem cells (CSCs), which are a small subset of cancer cells possessing robust stem cell properties within tumors. CSCs play crucial roles in tumor heterogeneity constitution, tumorigenesis, tumor relapse, metastasis, and resistance to anti-cancer therapies. Elucidation of how these CSCs maintain their stemness features is essential for the development of CSCs-based therapy. In this review, we summarize the present knowledge of intrinsic molecules and signaling pathways involved in hepatic CSCs, especially the CSC surface markers and associated signaling in regulating the stemness characteristics and the heterogeneous subpopulations within the CSC pool. In addition, we recapitulate the effects of crucial extrinsic cellular components in the tumor microenvironment, including stromal cells and immune cells, on the modulation of hepatic CSCs. Finally, we synopsize the currently valuable CSCs-targeted therapy strategies based on intervention in these intrinsic and extrinsic molecular mechanisms, in the hope of shedding light on better clinical management of HCC patients.

Aptamer-Functionalized Nanochannels for One-Step Detection of SARS-CoV-2 in Samples from COVID-19 Patients.

With the outbreak of COVID-19, which is fast transmitting and highly contagious, the development of rapid, highly specific, and sensitive detection kits has become a research hotspot. The existing assay methods for SARS-CoV-2 are mainly based on enzymatic reactions, which require expensive reagents, hindering popular use, especially in resource-constrained areas. Herein, we propose an aptamer-based method for the assay of SARS-CoV-2 via binding of the spike protein using functionalized biomimetic nanochannels. To get the analogous effect of human ACE2, a receptor for the spike protein, the aptamer to bind to the spike S1 protein has been first screened by a SELEX technique and then immobilized on the previously prepared nanochannels. In the presence of SARS-CoV-2, the changes in steric hindrance and charge density on the surface of the nanochannels will affect the ion transport, along with a rapid electrochemical response. Our method has been successfully applied to detect the viral particles in clinical pharyngeal swab specimens in one step without sample treatment. We expect this rapid, reagent-free, and sensitive assay method to be developed as a useful tool for diagnosing COVID-19.

Productive screening of single aptamers with ddPCR.

Antibodies have now been widely used for clinical treatment of a number of tumors. However, there are serious problems associated with antibody therapy, such as potential interactions of antibodies with the immune system as well as long production cycles. Recently, aptamers have been found to function similar to antibodies in terms of affinity and specificity to certain proteins and are attracting much attention for their low immunogenicity, easy chemical synthesis, and efficient penetration into tissues due to their small size. However, how to access high affinity and selectivity aptamers efficiently for further analysis is still open to be resolved. Herein, an aptamer discovery method that combines the continuous flow ddPCR technology with cytometer sorting of beads is reported, such that we have obtained DNA aptamers binding specifically to PD-1 with an affinity of over 60-fold higher than that for the best-reported method.

The emergence of the two cell fates and their associated switching for a negative auto-regulating gene.

BACKGROUND: Decisions in the cell that lead to its ultimate fate are important for fundamental cellular functions such as proliferation, growth, differentiation, development, and death. These cell fate decisions can be influenced by both the gene regulatory network and also environmental factors and can be modeled using simple gene feedback circuits. Negative auto-regulation is a common feedback motif in the gene circuits. It can act to reduce gene expression noise or induce oscillatory expression and is thought to lead to only one cell fate. Here, we present experimental and modeling data to suggest that a self-repressor circuit can lead to two cell fates under specific conditions. RESULTS: We show that the introduction of inducers capable of binding and unbinding to a self-repressing gene product (protein), thus regulating the associated gene, can lead to the emergence of two cell states. We suggest that the inducers can alter the effective regulatory binding and unbinding speed of the self-repressor regulatory protein to its destination DNA without changing the gene itself. The corresponding simulation results are consistent with the experimental findings. We propose physical and quantitative explanations for the origin of the two phenotypic cell fates. CONCLUSIONS: Our results suggest a mechanism for the emergence of multiple cell fates. This may explain the heterogeneity often observed among cell states, while illustrating that altering gene regulation strength can influence cell fates and their decision-making processes without genetic changes.

MED12 exerts an emerging role in actin-mediated cytokinesis via LIMK2/cofilin pathway in NSCLC.

BACKGROUND: Mediator complex subunit 12 (MED12) is an essential hub for transcriptional regulation, in which mutations and overexpression were reported to be associated with several kinds of malignancies. Nevertheless, the role of MED12 in non-small cell lung cancer (NSCLC) remains to be elucidated. METHODS: MED12 mutation was detected by Next-generation sequencing. The expression of MED12 in 179 human NSCLC tissue samples and 73 corresponding adjacent normal lung tissue samples was measured by immunohistochemistry (IHC). CRISPR-Cas9 was used to knock out MED12 in PC9 and SPC-A1 cells. MED12 rescued stable cell lines were generated by lentivirus infection. We traced cell division process by live cell imaging. The molecular mechanism of aborted cytokinesis resulted by MED12 knockout was investigated by RNA-seq. Effects of MED12 deletion on the proliferation of NSCLC cells were determined by MTT assay and Colony-formation assay in vitro and xenograft tumor model in nude mouse. Cell senescence was measured by SA-ß-gal staining. RESULTS: In our study, no MED12 exon mutation was detected in NSCLC samples, whereas we found that MED12 was overexpressed in human NSCLC tissues, which positively correlated with the tumor volume and adversely affected patient survival. Furthermore, knockout MED12 in NSCLC cell lines resulted in cytokinesis failure, displayed a multinuclear phenotype, and disposed to senescence, and become non-viable. Lack of MED12 decreased the proliferative potential of NSCLC cells and limited the tumor growth in vivo. Mechanism investigations revealed that MED12 knockout activated LIMK2, caused aberrant actin cytoskeleton remodeling, and disrupted the abscission of intercellular bridge, which led to the cytokinesis failure. Reconstitution of exogenous MED12 restored actin dynamics, normal cytokinesis and cell proliferation capacity in MED12 knockout cells. CONCLUSIONS: These results revealed a novel role of MED12 as an important regulator for maintaining accurate cytokinesis and survival in NSCLC cells, which may offer a therapeutic strategy to control tumor growth for NSCLC patients especially those highly expressed MED12.

Ceritinib Enhances the Efficacy of Substrate Chemotherapeutic Agent in Human ABCB1-Overexpressing Leukemia Cells In Vitro, In Vivo and Ex-Vivo.

BACKGROUND/AIMS: Multidrug resistance (MDR) triggered by ATP binding cassette (ABC) transporters, such as ABCB1, ABCC1, and ABCG2, is a key obstacle for successful cancer chemotherapy. There is currently no FDA-approved MDR modulator that can be used in clinic. Ceritinib, a selective ALK inhibitor, has been approved as the second-line treatment for ALK-positive non-small cell lung cancer. Here, we examined the role of ceritinib in leukemia associated MDR in therapy. METHODS: The cell proliferation was detected by MTT assay. The flow cytometry was used to detect the expression of cell surface protein and to detect the accumulation and efflux of rhodamine 123 (Rh123) or doxorubicin (Dox) in cells. The RT-PCR and Western blot were performed to detect the gene expression and protein expression levels, respectively. RESULTS: We found that ceritinib enhanced the efficacy of substrate chemotherapeutic agent in ABCB1-overexpressing K562/adr leukemia cells both in vitro and in vivo models, but neither in sensitive parental K562 leukemia cells nor in ABCC1-overexpressing HL-60/adr leukemia cells. Mechanistically, ceritinib significantly increased the intracellular accumulation of Rh123 or Dox but did neither alter ABCB1 expressions at both protein and mRNA levels nor block the phosphorylations of AKT and ERK1/2 at the concentration of MDR reversal. Importantly, ceritinib also increased the intracellular accumulation of Dox and enhanced the efficacy of Dox in primary leukemia cells in ex-vivo. CONCLUSION: Our results suggested that ceritinib enhanced the efficacy of substrate chemotherapeutic agent on inhibition of leukemia cell growth in vitro, in vivo and ex-vivo, which linked to block ABCB1 function, pumping out its substrate conventional chemotherapeutic agent, thereby increasing the intracellular accumulation. These suggest the combination of ceritinib and substrate chemotherapeutic drugs maybe an effective treatment of resistant leukemia patients with ABCB1-mediated MDR.

From molecules to cellular networks: past and future outlook.

Cellular networks have been the focus of studies in modern systems biology. They are crucial in understanding cell functions and related diseases. We review some past progress in both the theory and experiments, and we also provide several future perspectives for the field.

Prognostic value of total tumor volume in patients with nasopharyngeal carcinoma treated with intensity-modulated radiotherapy.

BACKGROUND: Few studies have evaluated the prognostic value of total tumor volume (TTV), which reflects both the primary tumor volume and nodal tumor volume, in NPC. Furthermore, the relationship between TTV and survival remains unknown. The purpose of this study was to evaluate the prognostic value of TTV in patients with NPC treated with intensity-modulated radiation therapy (IMRT). METHODS: TTV was retrospectively assessed in 455 patients with newly diagnosed, non-metastatic NPC. All patients were treated using IMRT; 91.1% (288/316) of patients with stage III-IVb also received cisplatin-based chemotherapy. Receiver operating characteristic (ROC) curves were used to identify the optimal TTV cut-off point and examine the prognostic value of combined TTV with current clinical stage. RESULTS: Mean TTV was 11.1 cm3 (range, 0.3-27.9 cm3) in stage I, 22.5 cm3 (1.3-92.4 cm3) in stage II, 40.6 cm3 in stage III (3.2-129.2 cm3), and 77.5 cm3 in stage IVa-b (7.1-284.1 cm3). For all patients, the 4-year estimated FFS, OS, DMFS, and LRRFS rates for patients with a TTV ≤ 28 vs. > 28 cm3 were 93 vs. 71.4% (P < 0.001), 95.1 vs. 75.4% (P < 0.001), 94.5 vs. 79.4% (P < 0.001), and 96.2 vs. 88% (P = 0.001). TTV was an independent prognostic factor for FFS, OS, DMFS and LRRFS in all patients. In stage III-IVb, 4-year estimated FFS, OS, DMFS, and LRRFS for a TTV ≤28 vs. >28 cm3 were 88.9 vs. 70.5% (P = 0.001), 96.2 vs. 72.7% (P < 0.001), 91.2 vs. 78.3% (P = 0.008), and 93.8 vs. 87.6% (P = 0.063). TTV was an independent prognostic factor for FFS, OS and DMFS in stage III-IVb. Receiver operating characteristic (ROC) curve analysis curves revealed adding TTV to clinical stage had superior prognostic value for treatment failure compared to clinical stage alone (P = 0.016). CONCLUSIONS: TTV is an important prognosticator for treatment outcome and significantly improves the prognostic value of the current staging system for patients with NPC treated with IMRT.

Developing a combined strategy for monitoring the progress of aptamer selection.

The development of a simple, low-cost, time-saving and universally applicable method to monitor the progression of aptamer selection is particularly challenging. Herein, a combined strategy dependent on quantitative polymerase chain reaction amplification curve (AC) and melting curve analysis (MCA) is developed to monitor the convergence of the aptamer species during selection progress. As a parallel and complementary method to affinity tests and binding analyses, the AC-MCA method can be used to achieve the DNA complexity assay when affinity is undetectable. It is independent of the target properties and SELEX methods. Therefore, it has been demonstrated as a universal monitoring tool in different SELEX methods towards different targets (small molecules, proteins, bacteria and cancer cells). The AC-MCA method will facilitate current new aptamer discovery and aptamer-based wide application.

Loss of ESRP2 Activates TAK1-MAPK Signaling through the Fetal RNA-Splicing Program to Promote Hepatocellular Carcinoma Progression.

Tumors usually display fetal-like characteristics, and many oncofetal proteins have been identified. However, fetal-like reprogramming of RNA splicing in hepatocellular carcinoma (HCC) is poorly understood. Here, it is demonstrated that the expression of epithelial splicing regulatory protein 2 (ESRP2), an RNA splicing factor, is suppressed in fetal hepatocytes and HCC, in parallel with tumor progression. By combining RNA-Seq with splicing analysis, it is identified that ESRP2 controls the fetal-to-adult switch of multiple splice isoforms in HCC. Functionally, ESRP2 suppressed cell proliferation and migration by specifically switching the alternative splicing (AS) of the TAK1 gene and restraining the expression of the fetal and oncogenic isoform, TAK1_ΔE12. Notably, aberrant TAK1 splicing led to the activation of p38MAPK signaling and predicted poor prognosis in HCC patients. Further investigation revealed that TAK1_ΔE12 protein interacted closely with TAB3 and formed liquid condensation in HCC cells, resulting in p38MAPK activation, enhanced cell migration, and accelerated tumorigenesis. Loss of ESRP2 sensitized HCC cells to TAK1 kinase inhibitor (TAK1i), promoting pyroptotic cell death and CD8+ T cell infiltration. Combining TAK1i with immune checkpoint therapy achieved potent tumor regression in mice. Overall, the findings reveal a previously unexplored onco-fetal reprogramming of RNA splicing and provide novel therapeutic avenues for HCC.

FASN Inhibition Decreases MHC-I Degradation and Synergizes with PD-L1 Checkpoint Blockade in Hepatocellular Carcinoma.

Immune checkpoint inhibitors (ICI) transformed the treatment landscape of hepatocellular carcinoma (HCC). Unfortunately, patients with attenuated MHC-I expression remain refractory to ICIs, and druggable targets for upregulating MHC-I are limited. Here, we found that genetic or pharmacologic inhibition of fatty acid synthase (FASN) increased MHC-I levels in HCC cells, promoting antigen presentation and stimulating antigen-specific CD8+ T-cell cytotoxicity. Mechanistically, FASN inhibition reduced palmitoylation of MHC-I that led to its lysosomal degradation. The palmitoyltransferase DHHC3 directly bound MHC-I and negatively regulated MHC-I protein levels. In an orthotopic HCC mouse model, Fasn deficiency enhanced MHC-I levels and promoted cancer cell killing by tumor-infiltrating CD8+ T cells. Moreover, the combination of two different FASN inhibitors, orlistat and TVB-2640, with anti-PD-L1 antibody robustly suppressed tumor growth in vivo. Multiplex IHC of human HCC samples and bioinformatic analysis of The Cancer Genome Atlas data further illustrated that lower expression of FASN was correlated with a higher percentage of cytotoxic CD8+ T cells. The identification of FASN as a negative regulator of MHC-I provides the rationale for combining FASN inhibitors and immunotherapy for treating HCC. SIGNIFICANCE: Inhibition of FASN increases MHC-I protein levels by suppressing its palmitoylation and lysosomal degradation, which stimulates immune activity against hepatocellular carcinoma and enhances the efficacy of immune checkpoint inhibition.

A self-amplifying plasmid based ultrasensitive biosensor for the detection of As(â ¢) in water.

Due to their advantages such as high sensitivity, good selectivity, low cost, easy preservation and strong environmental adaptability, microbial whole-cell biosensors have broad potential and application prospects in the detection and prevention of environmental pollutants. However, it is often restricted because the sensitivity and the limit of detection (LOD), are not enough to meet the actual detection demand. Here, we developed a novel biosensor signal-amplifier by introducing a replication protein of RepL, which is mainly based on the plasmid copy number inducible system. The new amplifier is applied to develop an As(Ⅲ) microbial sensor. Through the further optimization of regulatory elements, the sensor exhibits fast response, high sensitivity, low LOD and good linearity. The results show that our sensor of pMT012 has a LOD as low as 0.018 ppb, and a fast response with a response time of 10 and 40 min at 5 and 0.5 ppb As(Ⅲ), respectively. The excellent performance of the sensor not only enables us to achieve the detection rapidly and accurately, but also presents great potential applications for rapid determination of As(Ⅲ) acute toxicity of the pollutants. Meanwhile, this new signal-amplifier is universal and can be widely applied to many other biosensors.

Semi-automated and efficient parallel SELEX of aptamers for multiple targets.

In this study, we constructed and optimized a semi-automatic instrument to perform aptamer SELEX targeting multiple proteins simultaneously. Our work provides a simple SELEX platform characterized by real-time feedback, which is time efficient and can reduce human intervention. A number of aptamers were rapidly screened by this method. Moreover, the binding affinities of these aptamers were verified by various methods, including SPR and flow cytometry, which supports the applicability and reliability of our newly established aptamer SELEX system.

Graphene Based Nanohybrid Aptasensors in Environmental Monitoring: Concepts, Design and Future Outlook.

In view of ever-increasing environmental pollution, there is an immediate requirement to promote cheap, multiplexed, sensitive and fast biosensing systems to monitor these pollutants or contaminants. Aptamers have shown numerous advantages in being used as molecular recognition elements in various biosensing devices. Graphene and graphene-based materials/nanohybrids combined with several detection methods exhibit great potential owing to their exceptional optical, electronic and physicochemical properties which can be employed extensively to monitor environmental contaminants. For environmental monitoring applications, aptamers have been successfully combined with graphene-based nanohybrids to produce a wide range of innovative methodologies. Aptamers are immobilized at the surface of graphene based nanohybrids via covalent and non-covalent strategies. This review highlights the design, working principle, recent developmental advances and applications of graphene based nanohybrid aptasensors (GNH-Apts) (since January 2014 to September 2021) with a special emphasis on two major signal-transduction methods, i.e., optical and electrochemical for the monitoring of pesticides, heavy metals, bacteria, antibiotics, and organic compounds from different environmental samples (e.g., water, soil and related). Lastly, the challenges confronted by scientists and the possible future outlook have also been addressed. It is expected that high-performance graphene-based nanohybrid aptasensors would find broad applications in the field of environmental monitoring.

Customization of aptamer to develop CRISPR/Cas12a-derived ultrasensitive biosensor.

The CRISPR/Cas systems have provided wide biosensing applications. Particularly, the aptamer-involved CRISPR/Cas sensor system powerfully expanded to non-nucleic-acid targets. However, tailoring the sequence of the aptamer to explore the relationship between affinity and the activation of CRISPR/Cas12a trans-cleavage activity has not been reported yet. Herein, we developed a series of new aptamers toward the spike protein 1(S1) of SARS-CoV-2. Surface plasmon resonance measurements showed that the affinity of these aptamers to S1 was at the nM level. Subsequently, a "SET" effect (Sequence Essential Trans-cleavage activity) is discovered for the activation of CRISPR/Cas12a trans-cleavage activity. That is, an aptamer, as the activator, sequence needs to be tailored to activate CRISPR/Cas12a efficiently. A balance should be reached between affinity and activation ability. On the one hand, high affinity ensures target recognition performance, and on the other hand, activation can achieve adequate amplification and output of recognition signals. The optimized sequence (with 27 nucleotides, for short 27-nt) not only recognizes the target with a high affinity and specificity but also can trigger the CRISPR/Cas12a trans-cleavage activity efficiently, showing an excellent detection performance in electrochemical biosensors. The detection limit for SARS-CoV-2 S1 can be low at 1.5 pg mL-1. The new CRISPR/Cas12a-derived aptasensor also displays a remarkable ability to detect Beta, Delta, and Omicron variants but is selective toward other kinds of proteins. Above all, it is robust for point-of-care testing (POCT) in complex biological fluids, such as saliva, urine, and serum, and provides a universal and scalable detecting platform. Our results provide new insights into aptamer development and a different strategy for COVID-19 antigen detection and biosensor development.