The ultrasensitive measurement of protein markers plays a pivotal role in the early diagnosis of infectious and progressive diseases. Recently, digital methods such as those enabled by the Simoa Planar Array technology (SP-X System) have made significant progress in reaching ultrasensitive detection with clinically relevant protein biomarkers. The elicited Simoa technology is based on printing high-density capturing antibodies layers on the bottom of the wells of a microtiter plate, followed by a standard sandwich-type immunometric chemiluminescent detection. Such assay, reaching limit-of-detections (LODs) in the low femtomolar range, can be conveniently customized. An optimized Simoa SP-X assay for detecting and quantifying immunoglobulin M (IgM, non-specific indicator of inflammation) is developed herein and optimized. A full factorial experimental design is undertaken to optimize the assay, leading to a reduced experimental effort and increased quality of the information obtained concerning the traditional one-variable-at-a-time approach. The optimization process leads to an IgM LOD of 4 fm that compares well with those achieved with commercially available Simoa Planar Array kits. Remarkably, depositing both the capturing and detecting layer from a solution (0.1 μg mL −1 ) one order of magnitude less concentrated than in standard kits is needed, and the assay's cost is sizably reduced.

Early diagnosis and efficient treatments of oncological, neurological, inflammatory, and infectious diseases rely more and more on ultrasensitive detection of protein markers; the ultimate limit is a reliable immunoassay capable of single‐protein detection. Among protein biomarkers, cytokines play a key role in clinical diagnosis as they are involved in developing many complex diseases and disorders, such as chronic inflammatory diseases including metabolic syndrome, neurodegenerative diseases, and cardiovascular diseases, along with autoimmune diseases and cancer. Herein, the improvement of a Single Molecule with Transistor (SiMoT) is reported based on an electrolyte‐gated organic field‐effect transistor applied for the detection of cytokine IL‐6 in blood serum, reaching a limit‐of‐detection (LOD) of 1 ± 1 protein in a sample of 0.1 mL. The analytical performance levels are benchmarked against the Simoa Planar Array SP‐X technology, a benchtop chemiluminescent array. After comprehensive optimization, Simoa SP‐X by using a multivariate experimental design approach exhibits a LOD 103 higher than SiMoT. The proposed SiMoT electronic assay is label‐free, fast (30 min), and selective, paving the way for an ultra‐sensitive point‐of‐care immunoassay platform enabling pre‐symptomatic disease diagnosis.

This review discloses the technological advances involving enzyme‐based amperometric biosensors engaging challenging limits of detection as low as a single molecule. At first, we summarise the most recent findings concerning electrode modification toward the enhancement of the enzyme loading accomplished mainly through the deposition of nanomaterials. The increase of the electron transfer (ET) rate is mostly based on the enzyme site‐specific immobilization through the analysis of the enzyme structure/sequence and protein bioengineering is overviewed. However, both approaches are not appropriate to develop enzyme‐based amperometric biosensors able to reach reliable analytical detections below micro‐/nano‐molar. The last part is devoted to single‐molecule electrochemistry that has been widely exploited as a near‐field approach in the last decades as a proof‐of‐concept for the detection of single ET events. Organic electrochemical transistors operated as Faradaic current amplifiers do not detect below micro‐/nano‐molar. We here propose an alternative approach based on the combination of an electrochemical cell with a bipolar junction transistor in the extended base configuration, drawing some conclusions and future perspectives on the detection of single ET events at a large electrode for the development of Point‐of‐Care devices.

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The uniformity and long-term stability of physisorbed antibodies against SARS-CoV-2-Spike1 at a solid interface are addressed. High-performance sensing is accomplished, outmatching the analytical performance achieved with the chemical bonding of capture antibodies.