Signal Amplification Research Articles

Deep eutectic solvent-assisted synthesis of CeO2/CuO nanozymes for polymer dots integrated ratiometric fluorescence detection of glutathione

The current designs of ratiometric fluorescence sensing strategies primarily make use of the opposite responses of analyte target to two fluorescence probes, often overlooking their potential capabilities for signal amplification. Herein, a novel ratiometric fluorescence sensor was constructed for sensitive detection of glutathione (GSH) by combining CeO2/CuO nanozymes with poly[2,7-(9,9′-dioctylfluorene)-alt-4,7-bis(thiophen-2-yl) benzo-2,1,3-thiadiazole] (PFO-DBT)-based polymer dots (Pdots). The CeO2/CuO nanozymes with high oxidase-like activity were prepared via a deep eutectic solvent (DES) strategy and used for catalytic oxidization of o-phenylenediamine to produce fluorescent 2,3-diaminophenazine (DAP), which concurrently quenched the fluorescence of Pdots through photoinduced electron transfer (PET), thus amplifying the ratiometric signal. Upon addition of target GSH, the oxidase-like activity of CeO2/CuO were inhibited due to its blocking to metal active sites and scavenging superoxide anion (O2‧−), which responsively decreased the amount of produced DAP, and thus increased the fluorescence of Pdots. The designed CeO2/CuO-OPD-Pdots system showed a linear ratiometric response to GSH concentration within the range of 2 μM to 20 μM, with a limit of detection (LOD) determined as 1.24 μM. The proposed method could be used for selective detection of GSH in serum samples, and thus possessed potential application in clinic analysis.

Nanopore single molecule ATP detection based on the dual effects of specific capturing and signal amplification

Adenosine triphosphate (ATP) plays a crucial role in energy metabolism. Some serious diseases such as Alzheimer’s disease are related to abnormal ATP concentrations. Fast, sensitive, and specific ATP detection is important and challenging in disease diagnosis and biomedical research. In present study, conical nanopore was designed and employed to detect ATP at single molecule level. For overcoming the inherent defects such as non-selectivity and low sensitivity of nanopore in sensing small target molecule, two collaborative improvements of specific identification and signal amplification were proposed. We first used split aptamer capture probes (H1 and S1) to anchor ATP molecule, and then used auxiliary probes (H2 and H3) to form a long-nicked duplex ATP-DNA concatemer by hybridization chain reaction (HCR). Referring ATP-DNA concatemer rather than native ATP to nanopore sensor, characteristic current signals with high signal-to-noise ratio were obtained. By such indirect way, specific and sensitive ATP detection was realized with a linear range from 20 pM to 5 nM and a detection limit of 3.9 pM (S/N = 3). Additionally, the sensor exhibited many merits such as simplify, anti-interference, and especially allowing ATP detection in real sample. So, we believe that the nanopore sensor has good application prospects in screening and management of ATP-related diseases.

Generalized ratiometric surface-enhanced Raman scattering biosensor for okadaic acid in food based on Au-triggered signal amplification

BackgroundReliability and robustness have been recognized as key challenges for Surface-enhanced Raman scattering (SERS) analytical techniques. Quantifying the concentration of an analyte using a single characteristic peak from SERS has been a controversial topic because the Raman signal is susceptible to highly concentrated electromagnetic hotspots, inhomogeneity of SERS substrate, or non-standardization of measurement conditions. Ratiometric SERS strategies have been demonstrated as a promising solution to effectively balance and compensate for signal fluctuations caused by matrix heterogeneity. However, it is not easy to construct ratiometric SERS sensors with monitoring the ratio of two different signal intensities for target analysis. ResultsAn attempt has been made to develop a novel ratiometric biosensor that can be applied to detect okadaic acid (OA). Aptamer-anchored magnetic particles were first combined with gold-tagged short complementary DNA (Au-cDNA) to create heterogeneous nanostructures. When the target was present, the Au-cDNA was dissociated from nanostructures, and 4-nitrothiophenol (4-NTP) was initiated to reduce to 4-aminothiophenol (4-ATP) in the presence of hydrogen sources. The SERS ratio change of 4-NTP and 4-ATP was finally detected by AuNPs-coated film. OA was successfully quantified, and the detection limit was as low as 2.4524 ng/mL. The constructed biosensor had good stability and reproducibility with a relative standard deviation of less than 4.47%. The proposed method used gold nanoparticles as an intermediate to achieve catalytic signal amplification and subsequently increased the sensitivity of the biosensor. Significance and noveltyCatalytic reaction-based ratiometric SERS biosensors combine the multiple advantages of catalytic signal amplification and signal self-calibration and provide new insights into the development of stable, reproducible, and reliable SERS detection techniques. This ratiometric SERS technique offered a universal method that is anticipated to be applicable for the detection of other targets by substituting the aptamer.

Interface‐Driven DNA/Metal–Organic Framework Hybrids for Biosensing and Biomedical Applications

AbstractMetal–organic frameworks (MOFs) have emerged as promising materials for biosensing and biomedical applications due to their exceptional structural properties and tunable functionalities. Integrating functional DNA with MOFs offers numerous advantages, including enhanced colloidal stability, target‐induced signal amplification, improved cellular uptake, and controlled drug release. This focused review aims to highlight key insights into the DNA–MOF interface to facilitate the design and application of these conjugates. A succinct summary of the interactions between DNA and MOFs, covering both noncovalent adsorption and covalent conjugation is provided. Moreover, recent advancements in utilizing DNA–MOF conjugates in biosensors, with a specific emphasis on fluorescent and electrochemical sensing are discussed. Additionally, applications in bioimaging, controlled drug delivery, and nucleic acid delivery are explored. Finally, the limitations of existing designs and provide insightful perspectives for the future development of DNA–MOFs, aiming to expedite their integration and impact in the realm of biomedical applications are addressed.

Ultrasensitive and Rapid Circulating Tumor DNA Liquid Biopsy Using Surface-Confined Gene Amplification on Dispersible Magnetic Nano-Electrodes.

Circulating tumor DNA (ctDNA) detection has been acknowledged as a promising liquid biopsy approach for cancer diagnosis, with various ctDNA assays used for early detection and treatment monitoring. Dispersible magnetic nanoparticle-based electrochemical detection methods have been proposed as promising candidates for ctDNA detection based on the detection performance and features of the platform material. This study proposes a nanoparticle surface-localized genetic amplification approach by integrating Fe3O4-Au core-shell nanoparticles into polymerase chain reactions (PCR). These highly dispersible and magnetically responsive superparamagnetic nanoparticles act as nano-electrodes that amplify and accumulate target ctDNA in situ on the nanoparticle surface upon PCR amplification. These nanoparticles are subsequently captured and subjected to repetitive electrochemical measurements to induce reconfiguration-mediated signal amplification for ultrasensitive (∼3 aM) and rapid (∼7 min) metastatic breast cancer ctDNA detection in vitro. The detection platform can also detect metastatic biomarkers from in vivo samples, highlighting the potential for clinical applications and further expansion to rapid and ultrasensitive multiplex detection of various cancers.

Integration of high-entropy nanozyme and hollow In2S3 nanotube heterostructures decorated with WO3 for ultrasensitive PEC aptasensing of highly toxic mycotoxin

As a highly toxic mycotoxin, patulin threatens human and animal health. Herein, a new signal amplification strategy was designed for photoelectrochemical (PEC) analysis of patulin based on nanotube-like In2S3/WO3 heterostructures with the assistance of PtRuFeCoW high-entropy alloy nanorods (HEANRs). Specifically, the prominent photoactivity of the In2S3/WO3 was investigated by UV−vis diffuse reflectance and photoluminescence spectroscopy, coupled by elaborating the photoelectron transport mechanism in detail. Simultaneously, the excellent peroxidase-like activity of the PtRuFeCoW HEANRs was certificated mainly by UV–vis absorption spectroscopy. On such foundation, the PtRuFeCoW HEANRs modified capture DNA was cast onto the In2S3/WO3 based photoanode for catalyzing 4-chloro-1-naphthol (4-CN) oxidation to form insoluble benz-4-chloro-hexadienone (4-CD) precipitate in the presence of H2O2, ultimately weakening the PEC currents. The resulting PEC sensor exhibited a wider detection range from 0.1 pg mL−1 to 500 ng mL−1 and a lower detection limit was down to 0.063 pg mL−1 (S/N = 3). The HEANRs nanozyme assisted PEC method holds great promise for ultrasensitive detection of environmental pollutants in practice.

A novel signal amplified sandwich aptamer-based visual lateral flow assay based on self-assembly bifunctional nucleic acid for rapid and sensitive detection of CKMB

At present, rapid and sensitive detection of disease biomarkers is still the focus of early clinical diagnosis. As a rapid response to point-of-care testing (POCT), lateral flow assay (LFA) has been given serious attention. Unfortunately, due to the sensitivity limitations, LFA, especially AuNPs-based LFA, was difficult to play an important role in the detection of trace biomarkers. The combination of different signal amplification methods increases the complexity of operation and use, or prolongs the detection time, which sacrifices the advantages of fast and convenient of LFA. Hence, a novel sandwich aptamer-based visual LFA based on bifunctional nucleic acid (bFNA-LFA) was proposed for rapid and sensitive detection of creatine kinase MB (CKMB) in this study. Based on hybridization chain reaction (HCR) and nucleic acid hybridization, a bifunctional nucleic acid with target recognition and signal amplification was prepared, which effectively improved the sensitivity of CKMB detection without increasing the reaction time and depending on the detecting equipment. Under optimal reaction conditions, the limit of detection of 7.92 ng/mL was observed, which was 20 times lower than that of sandwich aptamer-based AuNPs LFA in the previous study. The proposed bFNA-LFA assay expanded a new idea in improving the sensitivity of LFA, and provided a reference for the subsequent application of nucleic acid nanomaterials in the detection of protein biomarkers.

Triple signal amplification electrochemical sensing platform for Hg2+ in water without direct modification of the working electrode

Triple signal amplification electrochemical sensing platform for Hg2+ in water without direct modification of the working electrode

Tuning quantum dots emission on DNA tetrahedron/silica nanosphere/graphene oxide nanointerface for ratiometric fluorescence assay of Pb2+ in multiplex samples

BackgroundAssembling framework nucleic acid (FNA) nanoarchitectures and tuning luminescent quantum dots (QDs) for fluorescence assays represent a versatile strategy in analytical territory. Rationally, FNA constructs could offer a preferential orientation to efficiently recognize the target and improve detection sensitivity, meanwhile, regulating size-dependent multicolor emissions of QDs in one analytical setting for ratiometric fluorescence assay would greatly simplify operation procedures. Nonetheless, such FNA/QDs-based ratiometric fluorescence nanoprobes remain rarely explored. ResultsWe designed a sensitive and signal amplification-free fluorescence aptasensor for lead ions (Pb2+) that potentially cause extensive contamination to environment, cosmetic, food and pharmaceuticals. Red and green emission CdTe quantum dots (rQDs and gQDs) were facilely prepared. Moreover, silica nanosphere encapsulating rQDs served as quantitative internal reference and scaffold to anchor a predesigned FNA and DNA sandwich containing Pb2+ binding aptamer and gQD modified DNA signal reporter. On binding of Pb2+, the gQD-DNA signal reporter was set free, resulting in fluorescence quenching at graphene oxide (GO) interface. Owing to the rigid structure of FNA, the fluorescence signal reporter orderly arranged at the silica nanosphere could sensitively respond to Pb2+ stimulation. The dose-dependent fluorescence signal-off mode enabled ratiometric analysis of Pb2+ without cumbersome signal amplification. Linear relationship was established between fluorescence intensity ratio (I555/I720) and Pb2+ concentration from 10 nM to 2 μM, with detection limit of 1.7 nM (0.43 ppb), well addressing the need for Pb2+ routine monitoring. The designed nanoprobe was applied to detection of Pb2+ in soil, cosmetic, milk, drug, and serum samples, with the sensitivity comparable to conventional ICP-MS technique. SignificanceGiven the programmable design of FNA and efficient recognition of target, flexible tuning of QDs emission, and signal amplification-free strategy, the present fluorescence nanoprobe could be a technical criterion for other heavy metal ions detection in a straightforward manner.

Fluorescence Sensing of Eclampsia Biomarkers via the Immunosorbent Atom Transfer Radical Polymerization Assay.

Accurate and quantitative detection of pre-eclampsia markers is crucial in reducing pregnancy mortality rates. This study introduces a novel approach utilizing a fluorescent biosensor by the immunosorbent atom transfer radical polymerization (immuno-ATRP) assay to detect the pre-eclampsia protein marker CD81. The critical step used in this sensor is the novel signal amplification strategy of fluorescein polymerization mediated by ferritin-enhanced controlled radical polymerization, which combines with a traditional enzyme-linked immunosorbent assay (ELISA) to further reduce the detection limit of the CD81 protein concentration. The fluorescence intensity was linear versus logarithmic CD81 protein concentration from 0.1 to 10,000 pg mL-1, and the detection limit was 0.067 pg mL-1. Surprisingly, in 30% normal human serum (NHS), the sensor can also detect target protein over 0.1-10,000 pg mL-1, with 0.083 pg mL-1 for the detection limit. Moreover, the proposed biosensor is designed to be cost-effective, making it accessible, particularly in resource-limited settings where expensive detection techniques may not be available. The affordability of this method enables widespread screening and monitoring of preeclampsia, ultimately benefiting many pregnant women by improving their healthcare outcomes. In short, developing of a low-cost and susceptible direct detection method for preeclampsia protein markers, such as CD81, through the use of the immuno-ATRP assay, has significant implications for reducing pregnancy mortality. This method holds promise for early detection, precise treatment, and improved management of preeclampsia, thereby contributing to better maternal and fetal health.

Optimizing the performance of silica nanoparticles functionalized with a near-infrared fluorescent dye for bioimaging applications

Modified fluorescent nanoparticles continue to emerge as promising candidates for drug delivery, bioimaging, and labeling tools for various biomedical applications. The ability of nanomaterials to fluorescently label cells allow for the enhanced detection and understanding of diseases. Silica nanoparticles have a variety of unique properties that can be harnessed for many different applications, causing their increased popularity. In combination with an organic dye, fluorescent nanoparticles demonstrate a vast range of advantageous properties including long photostability, surface modification, and signal amplification, thus allowing ease of manipulation to best suit bioimaging purposes. In this study, the Stöber method with tetraethyl orthosilicate (TEOS) and a fluorescent dye sulfo-Cy5-amine was used to synthesize fluorescent silica nanoparticles. The fluorescence spectra, zeta potential, quantum yield, cytotoxicity, and photostability were evaluated. The increased intracellular uptake and photostability of the dye-silica nanoparticles show their potential for bioimaging.

Dual structure-switching aptamer-mediated signal amplification cascade for SARS-CoV-2 detection

Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) at the end of 2019, the spread of the virus has posed a significant threat to public health and the global economy. This work proposed a one-step, dual-structure-switching aptamer-mediated signal amplification cascade for rapid and sensitive detection of the SARS-CoV-2 nucleocapsid protein. This system consisted of two DNA aptamers with structure-switching functionality and fuel DNA, where a cascade of strand hybridization and displacement triggered fluorescence generation and signal amplification. This aptamer-based amplification cascade required neither an amplification stage using enzymes nor pre-processing steps such as washing, viral isolation, and gene extraction. The assay could distinguish SARS-CoV-2 from other respiratory viruses and detect up to 1.0 PFU/assay of SARS-CoV-2 within 30 min at room temperature. In 35 nasopharyngeal clinical samples, the assay accurately assessed 25 positive and 10 negative clinical swab samples, which were confirmed using quantitative polymerase chain reaction. The strategy reported herein can help detect newly emerging pathogens and biomarkers of various diseases in liquid samples. In addition, the developed detection system consisting of only DNA and fluorophores can be widely integrated into liquid biopsy platforms for disease diagnosis.

On the relevance of weak measurements in dissipative quantum systems

We investigate the impact of dissipation, including energy relaxation and decoherence, on weak measurements. While weak measurements have been successful in signal amplification, dissipation can compromise their usefulness. More precisely, we show that in systems with a unique steady state, weak values always converge to an expectation value of the measured observable as dissipation time tends to infinity, in contrast to systems with multiple steady states, where the weak values can remain anomalous, i.e. outside the range of eigenvalues of the observable, even in the limit of an infinite dissipation time. In addition, we propose a method for extracting information about the dissipative dynamics of a system using weak values at short dissipation times. Specifically, we explore the amplification of the dissipation rate in a two-level system and the use of weak values to differentiate between Markovian and non-Markovian dissipative dynamics. We also find that weak measurements operating around a weak atom-cavity coupling can probe the atom dissipation through the weak value of non-Hermitian operators within the rotating-wave approximation of the weak interaction.

Functional Interface for Glycoprotein Sensing: Focusing on Biosensors.

Glycosylated proteins or glycoproteins make up a large family of glycoconjugates, and they participate in a variety of fundamental biological events. Glycoproteins have become important biomarkers in the diagnosis and treatment of a number of tumors. Biosensors are quite suitable for glycoprotein detection. The design and fabrication of a functional sensing interface play a crucial role in the biosensor construction to target glycoproteins. The functional interface, particularly receptors, typically determines the key characteristics of a biosensor, such as selectivity and sensitivity. Antibody, peptide, aptamer, boronic acid derivative, lectin, and molecularly imprinted polymer are all capable receptors for glycoprotein recognition, and each of these will be discussed. Most glycoproteins exist in low abundance, thus rendering signal amplification techniques indispensable. Nucleic acid-mediated and nanomaterial-mediated signal amplification for the detection of glycoproteins will be focused on herein. This review aims to highlight these different functional interfaces for glycoprotein sensing.

Colorimetric and Photothermal Dual-Modal Switching Lateral Flow Immunoassay Based on a Forced Dispersion Prussian Blue Nanocomposite for the Sensitive Detection of Prostate-Specific Antigen.

Prostate-specific antigen (PSA) is a key marker for a prostate cancer diagnosis. The low sensitivity of traditional lateral flow immunoassay (LFIA) methods makes them unsuitable for point-of-care testing. Herein, we designed a nanozyme by in situ growth of Prussian blue (PB) within the pores of dendritic mesoporous silica (DMSN). The PB was forcibly dispersed into the pores of DMSN, leading to an increase in exposed active sites. Consequently, the atom utilization is enhanced, resulting in superior peroxidase (POD)-like activity compared to that of cubic PB. Antibody-modified DMSN@PB nanozymes serve as immunological probes in an enzymatic-enhanced colorimetric and photothermal dual-signal LFIA for PSA detection. After systematic optimization, the LFIA based on DMSN@PB successfully achieves a 4-fold amplification of the colorimetric signal within 7 min through catalytic oxidation of the chromogenic substrate by POD-like activity. Moreover, DMSN@PB exhibits an excellent photothermal conversion ability under 808 nm laser irradiation. Accordingly, photothermal signals are introduced to improve the anti-interference ability and sensitivity of LFIA, exhibiting a wide linear range (1-40 ng mL-1) and a low PSA detection limit (0.202 ng mL-1), which satisfies the early detection level of prostate cancer. This research provides a more accurate and reliable visualization analysis methodology for the early diagnosis of prostate cancer.

Amplified Circularly Polarized Luminescence Behavior in Chiral Co-assembled Liquid Crystal Polymer Films via the Strategic Manipulation of Chiral Inducers.

One of the most important factors for the future application of circularly polarized luminescence (CPL) materials is their high dissymmetry factors (gem), and more and more studies are working tirelessly to focus on increasing the gem value. Herein, we chose an achiral liquid crystal polymer (LC-P) and two chiral binaphthyl-based inducers (R/S-3 and R/S-6) with different substitution positions (3,3' positions for R/S-3 and 6,6' positions for R/S-6) to construct chiral co-assemblies and explored their induced amplification CPL behaviors. Interestingly, after the thermal annealing treatment, this kind of chiral co-assembly (R/S-3)0.05-(LC-P)0.95 can emit a superior CPL signal (|gem| = 0.31 and λem = 424 nm), which achieves about 13-fold signal amplification in the spin-coated film, compared to (R/S-6)0.1-(LC-P)0.9 (|gem| = 0.023 and λem = 424 nm). This is because (R/S-3)0.05-(LC-P)0.95 could further co-assemble to form a more ordered arrangement LC state and generate regular helix nanofibers than that of (R/S-6)0.1-(LC-P)0.9. This work provides an efficient method for synthesizing high-quality CPL-active materials through the strategic manipulation of the structure of chiral binaphthyl-based inducers in chiral co-assembled LCP systems.

DNAzyme-driven tripedal DNA walker for ratiometric electrochemical aptasensor ultrasensitive detection of aflatoxin B1

Aflatoxin B1 is a potent carcinogen widely found in agricultural products and feed. Therefore, investigating the development of convenient, sensitive, and precise techniques for detecting AFB1 is critical. This work proposes a ratiometric electrochemical aptasensor based on DNAzyme-driven tripedal DNA walker for enzyme-free AFB1 detection. The design of multiple legs reduces the moving time on the electrode surface and enhances the value of signal response. In this approach, the tagging of ferrocene and methylene blue as signal switch molecules diminishes the impact of interferences and significantly enhances the reproducibility (RSD: 2.825%). Meanwhile, the tripedal DNA walker and DNAzyme dual signal amplification enabled the sensor to detect AFB1 traces in cereals. We achieved trace detection of AFB1 with a detection limit of 61 fg/mL and a wide linear range from 0.0001 to 10 ng/mL. Furthermore, the aptasensor success rate was comparable to that of HPLC-PMP. The results demonstrate enormous potential for detection. Furthermore, the method can detect various targets by altering the target aptamer, displaying attractive scalability. This suggests its tremendous prospective in detecting mycotoxins in food in the future.

Sensitive small extracellular vesicles associated circRNAs analysis combined with machine learning for precision identification of gastric cancer

Small extracellular vesicle associated circular RNAs (sEV-circRNAs) are emerging as promising biomarkers for gastric cancer diagnosis. Current research predominantly focuses on identifying these biomarkers through high-throughput sequencing. However, there has been insufficient exploration into the practical application of sEV-circRNAs for early gastric cancer diagnosis. In this study, we developed a sensitive electrochemical platform that leverages tetrahedron-Dox-AuNPs (TDA) tag and DNA tetrahedron-enhanced catalytic hairpin assembly (DT-CHA) to detect sEV-circRNAs. Based on the dual signal amplification of the TDA tag and DT-CHA, the platform can achieve low-concentration detection of the target, with a detection limit of 153.1 aM and a linear range from 1 fM to 1 nM. By profiling four sEV-circRNAs (circNRIP1, circRANGAP1, circCORO1C, and circSHKBP1) in a gastric cancer cohort and combining suitable ML diagnostic model, this platform performed well in distinguishing healthy donors from early GC patients. Thus, this confluence of a multi-biomarker approach with machine learning analysis, applied to plasma sEV-circRNAs, emerges as an important strategy for cancer screening.

Monomeric compounds from natural products for the treatment of pulmonary fibrosis: a review.

Pulmonary fibrosis (PF) is the end stage of lung injury and chronic lung diseases that results in diminished lung function, respiratory failure, and ultimately mortality. Despite extensive research, the pathogenesis of this disease remains elusive, and effective therapeutic options are currently limited, posing a significant clinical challenge. In addition, research on traditional Chinese medicine and naturopathic medicine is hampered by several complications due to complex compositionand lack of reference compounds. Natural product monomers, possessing diverse biological activities and excellent safety profiles, have emerged as potential candidates for preventing and treating PF. The effective anti-PF ingredients identified can be generally divided into flavonoids, saponins, polysaccharides, and alkaloids. Specifically, these monomeric compounds can attenuate inflammatory response, oxidative stress, and other physiopathological processes of the lung through many signaling pathways. They also improve pulmonary factors. Additionally, they ameliorate epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transdifferentiation (FMT) by regulating multiple signal amplifiers in the lungs, thereby mitigating PF. This review highlights the significant role of monomer compounds derived from natural products in reducing inflammation, oxidative stress, and inhibiting EMT process. The article provides comprehensive information and serves as a solid foundation for further exploration of new strategies to harness the potential of botanicals in the treatment of PF.

Proteolytic reaction-based versatile electrochemical biosensor chip for simple and quantitative point-of-care testing

Electrochemical protease biosensors are well-suited for simple and quantitative point-of-care testing. However, there has been limited development of versatile chip formats similar to lateral-flow and glucose test strips for electrochemical proteases biosensors. This paper presents a versatile electrochemical biosensor chip for the wash-free and sensitive detection of proteases. The chip comprises (i) a reaction well, (ii) micropatterned electrodes on a glass substrate, and (iii) target protease-dependent filter paper. All (bio)chemicals required for the proteolytic reaction and signal amplification were predried on filter paper, which was then placed inside the reaction well. Upon injecting the sample solution into the reaction well, most of the dried (bio)chemicals dissolved rapidly. Wash-free and sensitive protease detection was achieved using electrochemical-enzymatic (EN) redox cycling of a redox-active species (4-amino-1-naphthol, AN) liberated from a peptide substrate through a rapid proteolytic reaction. The EN redox cycling involves a working electrode, AN, glucose dehydrogenase, and glucose. To enhance dissolvability and stability, (bio)chemicals were dried using a surfactant and stabilizer and stored at 4 °C with vacuum sealing. To minimize the effect of background variation among the different samples, a high concentration of glucose (200 mM) was used, and the difference between the two charge values obtained before and after the incubation period was measured. The biosensor chips successfully detected three proteases: thrombin, leucine aminopeptidase (LAP), and 3CL protease from coronavirus SARS-CoV-2 (Mpro). The detection limits of thrombin in artificial serum, Mpro in artificial saliva, and LAP in human serum were approximately 100, 100 pg/mL, and 8 U/L, respectively.