(D) The background fluorescence of carboxylated MBs and (F) SA coated MBs

(D) The background fluorescence of carboxylated MBs and (F) SA coated MBs. microscope. The number of FL wells with MBs (FL wells number) and the number of wells with MBs (MBs wells number) were counted, respectively. The percentage of FL wells was calculated by dividing (FL wells number) by (MBs wells number). The higher the percentage of FL wells, the higher the N protein concentration. The detection limit of this digital method for N protein was 33.28?pg/mL, which was 300 times lower than traditional double-antibody sandwich based enzyme-linked immunosorbent assay (ELISA). Keywords: Digital detection, Microfluidic chip, COVID-19, SARS-CoV2, Nucleocapsid protein, Aptamer/antibody sandwich Graphical abstract An aptamer/antibody sandwich method for digital detection of SARS-CoV2 nucleocapsid protein based on microfluidic chip with femtoliter-sized wells was proposed for the first time. Open in a separate window 1.?Introduction The outbreak of coronavirus disease 2019 (COVID-19) caused a global health crisis. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), and it has high infectivity and high lethality [1]. More than 190 million people have been diagnosed with COVID-19, and millions died from COVID-19 [2]. Although COVID-19 vaccines have been administered worldwide, early diagnosis remains an effective way for early isolation of patients with COVID-19 and control the spread of SARS-CoV-2 in the context of the limited effectiveness of the vaccine and the fast mutations of SARS-CoV-2 [3]. At present, the reverse transcription-polymerase chain reaction (RT-PCR) test is the gold standard for the diagnosis of SARS-CoV-2 [4]. Nevertheless, there is growing evidence demonstrating that this technique may Fanapanel generate false-negative results [5]. Direct detection of host antibodies (IgM) from a small volume of serum or plasma is generally less sensitive than Fanapanel RT-PCR, and SARS-COV-2 may be detected within the first week after symptoms onset, while the viral load is typically higher. When detected 10C14 days after the onset of symptoms, the viral load is low Rabbit Polyclonal to BAGE3 or undetectable, the performance of these tests decreases significantly [6,7]. In addition to viral RNA and host Fanapanel antibodies, viral structural proteins are also alternative targets for SARS-CoV2 detection, such as nucleocapsid protein (N protein), spike protein (S protein), membrane protein, and envelope protein [8]. N protein is the most abundant protein in SARS-CoV2 and is highly conserved [9], and there are no homologous proteins in the human body. Therefore, N protein is an ideal biomarker for the early diagnosis of SARS-CoV2. Aptamers are high-affinity single-stranded DNA or RNA molecules to specific targets and screened using an in vitro selection procedure called Systematic Evolution of Ligands by EXponential enrichment (SELEX) discovered by Gold and Szostak in the 1990s [10]. Up till now, many nucleic acid aptamers have been identified using Fanapanel SELEX, including aptamers that are specific to pathogenic organisms [11], proteins [12], cells [13], heavy metal ions [14], and small molecules [15]. Compared with antibodies widely used in biomedicine, the aptamer is a novel molecular recognition element. The specificity and affinity of aptamer binding to target are Fanapanel equal to or even stronger than that of antibody. Furthermore, aptamer has the advantages of a short screening cycle, low immunogenicity, simple and rapid synthesis, low cost, easy modification, good stability and can be transported at room temperature [16]. After the outbreak of COVID-19, Zhaofeng Luo’s group quickly screened out the aptamers of N protein [17] and demonstrated that the antibody/aptamer sandwich method has a higher sensitivity for N protein detection than double-antibody or double-aptamer sandwich methods. The usage of aptamers also reduces the complexity and cost of the detection system. However, the detection sensitivity still needs to be improved if applied in early diagnosis. A digital assay is one in which the sample is partitioned into many containers so that each partition contains a discrete number of biological molecules. The digital assays, usually based on microfluidic compartmentalized techniques, such as digital enzyme-linked immunosorbent assay (dELISA) or digital PCR, bring.