Sensitive Biomarker Detection Research Articles

A Super-Antifouling Electrochemical Biosensor for Protein Detection in Complex Biofluids Based on PEGylated Multifunctional Peptide.

Overcoming the influence of interfering substances in the environment and achieving superior sensing performance are significant challenges in biomarker detection within complex matrices. Herein, an integrated electrochemical sensing platform for sensitive detection of biomarkers in complex biofluids was developed based on a newly designed PEGylated multifunctional peptide (PEG-MPEP). The designed PEG-MPEP contains a poly(serine) sequence (-ssssss-) as the antifouling part and recognition peptide sequence (-avwgrwh) specific for the target human immunoglobulin G (IgG). To improve the peptide stability to protease hydrolysis, d-amino acids were adopted to synthesize the whole peptide. Additionally, the PEGylation can further enhance the stability of the peptide, and the PEG itself was also antifouling, ensuring superstrong antifouling capability of the PEG-MPEP. The designed PEG-MPEP-based biosensor possessed a high sensitivity for the detection of IgG in the range of 1.0 pg mL-1 to 1.0 μg mL-1, with a low limit of detection (0.41 pg mL-1), and it was capable of assaying targets accurately in real serum samples. Compared with conventional peptide-modified biosensors, the PEG-MPEP-modified biosensor exhibited superior antifouling and antihydrolysis properties in complex biofluid, showcasing promising potential for practical assay applications.

Metal-ligand cross-link strategy engineered iron-doped dopamine-based superstructure as peroxidase-like nanozymes for detection of glucose.

Nanozymes are nanomaterials with mimetic enzyme properties and the related research has attracted much attention. It is of great value to develop methods to construct nanozymes and to study their application in bioanalysis. Herein, the metal-ligand cross-linking strategy was developed to fabricate superstructure nanozymes. This strategy takes advantage of beingeasy to operate, adjustable, cheap, and universal. The fabricated superstructure nanozymes possess efficient peroxidase-like catalytic activity. The enzyme reaction kinetic tests demonstrated that for TMB and H2O2, the Km is 0.229 and 1.308mM, respectively. Furthermore, these superstructure nanozymes are applied to highly efficient and sensitive detection of glucose. The linear range for detecting glucose is 20-2000μM, and the limit of detection is 17.5μM. Furthermore, mechanistic research illustrated that this integrated system oxidizes glucose to produce hydrogen peroxide and further catalyzes the production of ·OH and O2·-, which results in a chromogenic reaction of oxidized TMB for the detection of glucose. This work could not only contribute to the development of efficient nanozymes but also inspire research in the highly sensitive detection of other biomarkers.

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.

APPROACH: Sensitive Detection of Exosomal Biomarkers by Aptamer-Mediated Proximity Ligation Assay and Time-Resolved Förster Resonance Energy Transfer.

Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an "APPROACH" strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙μL-1, 0.77 ng∙μL-1, and 1.1 ng∙μL-1, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙μL-1 in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.

Modular probe integrating with quantum dots based versatile platform for sensitive and label-free biomarker detection

Multiplexed analysis of biomarkers in a single sample tube is essential for accurate diagnosis and therapy of diseases. However, the existing detection platforms suffer from many drawbacks, such as low specificity, limited applicable sceneries, and complicated operation. Hence, it is highly important to develop a versatile biomarker detection platform that can be used for disease diagnosis and pathophysiological research. In this study, we provide a versatile method for detecting biomarkers using dual-loop probes and quantum dots (QDs). This approach utilizes a dual-loop probe that consists of a recognition module for identifying specific targets, a template recognition module for initiating subsequent chain replacement cycles, and a signal module for facilitating the fixation of QDs on the 96-well plate. The lower limit of detection for miRNA-21 is determined to be at the aM level. Furthermore, this design may be easily expanded to simultaneously detect several targets, such as miRNA and C-reactive protein. The experimental results demonstrated the successful construction of the versatile biomarkers detection platform, and indicated that the sensitive and versatile platform has significant potential in the areas of bio-sensing, clinical diagnostics, and environmental sample analysis.

Correlation of salivary hs-CRP With conventional cardiovascular risk markers and hba1c in prediabetic subjects

Prediabetes has attracted considerable attention because it increases the risk of developing diabetes and cardiovascular disease. Prediabetes is also associated with obesity and dyslipidemia. Chronic low-grade inflammation is a crucial element in the clinical course of metabolic syndrome, and cardiovascular and cerebrovascular diseases. C-Reactive Protein (CRP) is a biomarker of inflammation that tends to increase in patients with diabetes and prediabetes. Most serological markers of diabetes are detected using invasive techniques that cause anxiety and pain. Therefore, the use of noninvasive techniques for frequent biomarker monitoring is necessary. Salivary diagnostics is an upcoming field for sensitive biomarker detection, such as hs-CRP. HbA1C is a valuable glycemic risk marker and has proven to be a reliable test of time. Both HbA1C and CRP play pivotal roles in diabetes and prediabetes. hs-CRP is a measure of CRP level with greater accuracy. The lower limit of its measurement is 0.01 mg/L and the measurement is more than100 times as sensitive than the CRP measurement (lower limit 5 mg/L). Therefore, our study aimed to evaluate and establish a relationship between these factors in prediabetics. Since metabolic syndrome and prediabetes are the cause and effect of each other, correlating anthropometric measurements and lipid profile with HbA1C and hs-CRP will be our focus.

Functionalized DNA Origami-Enabled Detection of Biomarkers.

Biomarkers are crucial physiological and pathological indicators in the host. Over the years, numerous detection methods have been developed for biomarkers, given their significant potential in various biological and biomedical applications. Among these, the detection system based on functionalized DNA origami has emerged as a promising approach due to its precise control over sensing modules, enabling sensitive, specific, and programmable biomarker detection. We summarize the advancements in biomarker detection using functionalized DNA origami, focusing on strategies for DNA origami functionalization, mechanisms of biomarker recognition, and applications in disease diagnosis and monitoring. These applications are organized into sections based on the type of biomarkers-nucleic acids, proteins, small molecules, and ions-and concludes with a discussion on the advantages and challenges associated with using functionalized DNA origami systems for biomarker detection.

Colorimetric detection of electrolyte ions in blood based on biphasic microdroplet extraction

BackgroundTimely diagnosis and prevention of diseases require rapid and sensitive detection of biomarkers from blood samples without external interference. Abnormal electrolyte ion levels in the blood are closely linked to various physiological disorders, including hypertension. Therefore, accurate, interference-free, and precise measurement of electrolyte ion concentrations in the blood is particularly important. ResultsIn this work, a colorimetric sensor based on a biphasic microdroplet extraction is proposed for the detection of electrolyte ions in the blood. This sensor employs mini-pillar arrays to facilitate contact between adjacent blood microdroplets and organic microdroplets serving as sensing phases, with any color changes being monitored through a smartphone's colorimetric software. The sensor is highly resistant to interference and does not require pre-treatment of the blood samples. Remarkably, the sensor exhibits exceptional reliability and stability, allowing for rapid enrichment and detection of K+, Na+, and Cl− in the blood within 10 s (Cl−), 15 s (K+) and 40 s (Na+) respectively. SignificanceThe colorimetric sensor based on biphasic microdroplet extraction offers portability due to its compact size and ease of operation without the need for large instruments. Additionally, it is location-independent, making it a promising tool for real-time biomarker detection in body fluids such as blood.

Electrospun EU/HPMC nanofibers decorated by ZIF-8 nanoparticle as the advanced electrochemical biosensor modifier for sensitive and selective detection of c-MET cancer biomarker in human plasma sample

This research presents a selective and sensitive electrochemical biosensor for the detection of the mesenchymal–epithelial transition factor (c-MET). The biosensing is based on a modification of the SPCE (screen-printed carbon electrode) with the electrospun nanofiber containing eudragit (EU), hydroxypropyl methylcellulose (HPMC), and Zeolite imidazolate frameworks (ZIF-8) nanoparticles. EU/HPMC/ZIF-8 nanofibers have presented a high capability of electron transfer, and more active surface area than bare SPCE due to synergistic effects between EU, HPMC, and ZIF-8. On the other hand, EU/HPMC nanofibers provided high porosity, flexible structures, high specific surface area, and good mechanical strength. The presence of ZIF-8 nanoparticles improved the immobilization of anti-c-MET on the modified SPCE and also resulted in increasing the conductivity. By c-MET incubation on the modified SPCE, c-MET was connected to anti-c-MET, and consequently the electrochemical signal of [Fe(CN)6]3-/4- as the anion redox probe was reduced. In order to investigate the structural and morphological characteristics and elemental composition of electrospun nanofibers, various characterization methods including FE-SEM, XRD, FTIR, and EDS were used. Under optimum conditions with a working potential range −0.3−0.6 V (vs. Ag/AgCl), linear range (LR), correlation coefficient (R2), sensitivity, and limit of detection (LOD) were acquired at 100 fg/mL-100 ng/mL, 0.9985, 53.28 μA/cm2.dec, and 1.28 fg/mL, respectively. Moreover, the mentioned biosensor was investigated in a human plasma sample to determine c-MET and showed ideal results including reproducibility, stability, and good selectivity against other proteins.

An electrochemiluminescent magneto-immunosensor for ultrasensitive detection of hs-cTnI on a microfluidic chip

Sensitive detection and precise quantitation of trace-level crucial biomarkers in a complex sample matrix has become an important area of research. For example, the detection of high-sensitivity cardiac troponin I (hs-cTnI) is strongly recommended in clinical guidelines for early diagnosis of acute myocardial infarction. Based on the use of an electrode modified by single-walled carbon nanotubes (SWCNTs) and a Ru(bpy)32+-doped silica nanoparticle (Ru@SiO2)/tripropylamine (TPA) system, a novel type of electrochemiluminescent (ECL) magneto-immunosensor is developed for ultrasensitive detection of hs-cTnI. In this approach, a large amount of [Ru(bpy)3]2+ is loaded in SiO2 (silica nanoparticles) as luminophores with high luminescent efficiency and SWCNTs as electrode surface modification material with excellent electrooxidation ability for TPA. Subsequently, a hierarchical micropillar array of microstructures is fabricated with a magnet placed at each end to efficiently confine a single layer of immunomagnetic microbeads on the surface of the electrode and enable 7.5-fold signal enhancement. In particular, the use of transparent SWCNTs to modify a transparent ITO electrode provides a two-order-of-magnitude ECL signal amplification. A good linear calibration curve is developed for hs-cTnI concentrations over a wide range from 10 fg/ml to 10 ng/ml, with the limit of detection calculated as 8.720 fg/ml (S/N = 3). This ultrasensitive immunosensor exhibits superior detection performance with remarkable stability, reproducibility, and selectivity. Satisfactory recoveries are obtained in the detection of hs-cTnI in human serum, providing a potential analysis protocol for clinical applications.

Recent progress in microRNA detection using integrated electric fields and optical detection methods

Low-cost, highly-sensitivity, and minimally invasive tests for the detection and monitoring of life-threatening diseases and disorders can reduce the worldwide disease burden. Despite a number of interdisciplinary research efforts, there are still challenges remaining to be addressed, so clinically significant amounts of relevant biomarkers in body fluids can be detected with low assay cost, high sensitivity, and speed at point-of-care settings. Although the conventional proteomic technologies have shown promise, their ability to detect all levels of disease progression from early to advanced stages is limited to a limited number of diseases. One potential avenue for early diagnosis is microRNA (miRNA). Due to their upstream positions in regulatory cascades, blood-based miRNAs are sensitive biomarkers that are detectable earlier than those targeted by other methods. Therefore, miRNA is a promising diagnostic biomarker for many diseases, including those lacking optimal diagnostic tools. Electric fields have been utilized to develop various biomedical assays including cell separation, molecules detection and analysis. Recently, there has been a great interest in the utility of electric fields with optical detection methods, including fluorescence and surface plasmons toward biomarker detection. This mini review first summarizes the recent development of miRNA as a biomarker. Second, the utility of electric fields and their integration with fluorescence detection methods will be discussed. Next, recent studies that utilized electric fields and optical detection methods will be discussed. Finally, in conclusion, technology gaps and improvements needed to enable low-cost and sensitive biomarker detection in point-of-care settings will be discussed.

A Panorama of Extracellular Vesicle Applications: From Biomarker Detection to Therapeutics.

Extracellular vesicles (EVs) secreted by all cell types are involved in the cell-to-cell transfer of regulatory factors that influence cell and tissue phenotypes in normal and diseased tissues. EVs are thus a rich source of biomarker targets for assays that analyze blood and urinary EVs for disease diagnosis. Sensitive biomarker detection in EVs derived from specific cell populations is a key major hurdle when analyzing complex biological samples, but innovative approaches surveyed in this Perspective can streamline EV isolation and enhance the sensitivity of EV detection procedures required for clinical application of EV-based diagnostics and therapeutics, including nanotechnology and microfluidics, to achieve EV characterizations. Finally, this Perspective also outlines opportunities and challenges remaining for clinical translation of EV-based assays.

ReS2-MoS2 heterojunction nanotube FET with superlattice structures for ultrasensitive detection of miRNA

Ultrasensitive detection of cancer biomarkers holds immense importance in facilitating the early diagnosis and treatment of cancer. Compared to conventional detection techniques, field-effect transistor (FET) biosensors have demonstrated substantial potential in achieving sensitive detection of cancer biomarkers. However, the ultrasensitive FET still remains great challenges due to the limited electron transport capacity and sensitivity of channel materials. Herein, we employed rhenium disulfide-molybdenum disulfide nanotube (ReS2-MoS2 NT) featuring superlattice structures as channel materials in FET biosensors for the highly sensitive detection of cancer biomarker miRNA-21. More concretely, ReS2-MoS2 NT featuring superlattice structures was synthesized using the anodic aluminum oxide (AAO) template sacrifice method and atomic layer deposition (ALD) method. The electrical properties of single ReS2-MoS2 NT FET were regulated by adjusting the total layers and number of heterojunction interfaces, and the optimal number of heterojunction interfaces of 7 was obtained. Finally, FET biosensors utilizing the network ReS2-MoS2 NT as the channel materials were fabricated and exhibited remarkable sensitivity in detecting miRNA-21 under an external light source. The linear range for detection spanned from 10 aM to 1 nM, with an exceptionally low detection limit of 2.1 aM. This study holds promise for replacing current channel materials and surpassing the detection threshold of cancer biomarkers in the future.

Visual and quantitative lateral flow immunoassay based on Au@PS SERS tags for multiplex cardiac biomarkers

Rapid and sensitive detection of multiple biomarkers by lateral flow immunoassay (LFIA) remains challenging for signal amplification for commonly used nanotags. Herein, we report a novel LFIA strip for visual and highly sensitive analysis of two cardiac biomarkers based on functionalized gold nanoparticles @ polystyrene microsphere (Au@PS）microcavity as surface-enhanced Raman scattering (SERS) tags. Antibody-modified Au@PS was designed as a SERS label. The evanescent waves propagating along the surface of the PS microcavity and the localized surface plasmons of the gold nanoparticles were coupled to enhance the light-matter interaction synergistically for Raman signal enhancement. In this strategy, the proposed Au@PS SERS tags-based LFIA was carried out to quantify the content of the heart failure and infarct biomarkers synchronously within 15 min and get the limits of detection of 1 pg/mL and 10 pg/mL for cardiac troponin I (cTnI) and N-terminal natriuretic peptide precursor (NT-proBNP), respectively. The results demonstrated 10–20 folds more sensitivity than that of the standard colloidal gold strip and fluorescent strip for the same biomarkers. This novel quantitative LFIA shows promise as a high-sensitive and visual sensing method for relevant clinical and forensic analysis.

Sensitive detection of multiple blood biomarkers via immunomagnetic exosomal PCR for the diagnosis of Alzheimer's disease.

Blood exosomes are emerging as potential biomarkers for diagnosing brain diseases such as Alzheimer's disease (AD). There is currently a lack of an ultrasensitive technology for identifying core AD biomarkers in blood exosomes to optimize the utility of biomarkers in clinical practice. Here, an immunomagnetic exosomal polymerase chain reaction (iMEP) platform was developed using DNA-conjugated antibodies for the rapid detection of amyloid-β (Aβ1-40 and Aβ1-42) and phosphorylated tau (p-tau396,404 and p-tau181) in clinical blood exosomes. The toehold shift-mediated DNA affinity pulldown eliminates the high detection background, which allows the detection of biomarkers at concentrations down to 10 femtograms per milliliter. With the iMEP assay, exosomal Aβ1-42 was more accurate in differentiating patients with AD from healthy individuals compared with exosomal p-tau181 and p-tau396,404, with a sensitivity of 95.0% and a specificity of 95.0%. The iMEP technique is also adept at quantifying the levels of different exosomal biomarkers associated with disease pathogenesis.

Abstract 7092: Cancer biomarker discovery with scalable biofluid proteomics

Abstract Biofluid proteomics opens opportunities for the discovery of early detection, diagnostic, and prognostic cancer biomarkers through non-invasive or minimally invasive screening. Plasma and serum are accessible sample types, however, the high dynamic range of proteins creates analytical challenges for the sensitive detection of potential biomarkers. In this work, we compare two proteomic approaches for large-scale cancer biomarker discovery: the Olink Proximity Extension Assay and the Seer Proteograph Product Suite. Olink Explore 3072 is an immunoassay that gains specificity by using dual antibody recognition to measure approx. 3,000 proteins in each sample. Antibody pairs are tagged with oligonucleotides, which hybridize and are amplified before measurement by next generation sequencing. Seer Proteograph is an enrichment strategy that uses a proprietary mix of functionalized magnetic nanoparticles with affinity for certain amino acids and functional groups in native state proteins. The resulting peptides are measured using untargeted mass spectrometry to identify proteins at the peptide level, paving the way for the identification of proteoforms and peptide variants. Aliquots of human plasma from 78 samples representing a range of cancer indications were obtained through the Discovery Life Sciences biobank and used for analysis by both the Olink and Proteograph workflows. Olink analysis achieved Pearson correlation scores of greater than or equal to 0.8 when correlating each of seven process controls to each other for all approx. 3000 proteins. On average, greater than or equal to 75% of the proteins were detected across all panels in all sample groups except for Normal and Benign groups (64% and 74% respectively). After stringent FDR filtering to ensure only high-quality data was analyzed, the Seer Proteograph workflow quantified 4,323 proteins across all 78 samples. Pearson correlation scores of greater than or equal to 0.96 were achieved when comparing digestion controls, process controls, and sample cleanup controls. Both platforms achieved detection of proteins across the dynamic range of plasma, including low abundance proteins. The Olink Explore 3072 and Seer Proteograph platforms were found to be complementary, with approx. 15% overlap in measured proteins. Cancer biomarker discovery at the proteomic level is bolstered by recent advances in analytical capabilities, as revealed by the Olink Explore and Seer Proteograph platforms. Importantly, complementary implementation of these two workflows will enable comprehensive biomarker analysis. Citation Format: Danielle B. Gutierrez, Jessica L. Moore, Jaison Arivalagan, Tiffany Louie, Leslie Wilkinson, Ray Clemens. Cancer biomarker discovery with scalable biofluid proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7092.

Ultra-stable and highly-bright CsPbBr3 perovskite/silica nanocomposites for miRNA detection based on digital single-nanoparticle counting

Single-nanoparticle counting (SNPC) based on fluorescent tag (FT) stands out for its capacity to achieve amplification-free and sensitive detection of biomarkers. The stability and luminescence of FT are important to the sensitivity and reliability of SPNC. In this work, we developed novel perovskite/silica nanocomposites by in-situ nanoconfined growth of CsPbBr3 nanocrystals inside mesoporous structure of silica nanoparticles. PbBr(OH) was formed in an alkaline-assisted reaction triggered by water on the surface of CsPbBr3 nanocrystals. The as-obtained nanocomposites, featuring dual protection from silica matrix and PbBr(OH), exhibited high absolute photoluminescence quantum yield (PLQY) of 86.5% and demonstrated outstanding PL stability confronting with water, heat, ultrasound and UV-irradiation, which is desired by SNPC-based biosensor. Thereafter, these nanocomposites were used to construct an operationally friendly SNPC assay for the amplification-free quantification of cancer-associated miRNA. Quantitative detection of miRNA could be accomplished by directly counting the number of nanocomposites using a flow cytometer in this assay. This strategy did not ask for multiple washing steps and demonstrated specific and sensitive detection of miRNA 21, which exhibited a dynamic range of 1–1000 pM and limit of detection of 79 amol. The employment of highly stable perovskite/silica nanocomposites improved the test reliability and stability of SNPC, revealing the vast potential of perovskites in biosensing.

Renin imprinted Poly(methyldopa) for biomarker detection and disease therapy

Conventional molecularly imprinted polymers (MIPs) perform their functions principally depended on their three dimensional (3D) imprinted cavities (recognition sites) of templates. Here, retaining the function of recognition sites resulted from the imprinting of template molecules, the role of functional monomers is explored and expanded. Briefly, a class of dual-functional renin imprinted poly(methyldopa) (RMIP) is prepared, consisting of a drug-type function monomer (methyldopa, clinical high blood pressure drug) and a corresponding disease biomarker (renin, biomarker for high blood pressure disease). To boost target-to-receptor binding ratio and sensitivity, the microstructure of recognition sites is beforehand calculated and designed by Density Functional Theory calculations, and the whole interfacial structure, property and thickness of RMIP film is regulated by adjusting the polymerization techniques. The dual-functional applications of RMIP for biomarker detection and disease therapy in vivo is explored. Such RMIP-based biosensors achieves highly sensitive biomarker detection, where the LODs reaches down to 1.31 × 10−6 and 1.26 × 10−6 ng mL−1 for electrochemical and chemical polymers, respectively, and the application for disease therapy in vivo has been verified where displays the obviously decreased blood pressure values of mice. No acute and long-term toxicity is found from the pathological slices, declaring the promising clinical application potential of such engineered RMIP nanostructure.

A point-of-care electrochemical biosensor for the rapid and sensitive detection of biomarkers in murine models with LPS-induced sepsis

Sepsis is a life-threatening condition, which is irreversible if diagnosis and intervention are delayed. The response of the immune cells towards an infection triggers widespread inflammation through the production of cytokines, which may result in multiple organ dysfunction and eventual death. Conventional detection techniques fail to provide a rapid diagnosis because of their limited sensitivity and tedious protocol. This study proposes a point-of-care (POC) electrochemical biosensor that overcomes the limitations of current biosensing technologies in the clinical setting by its integration with electrokinetics, enhancing the sensitivity to picogram level compared with the nanogram limit of current diagnostic technologies. This biosensor promotes the use of a microelectrode strip to address the limitations of conventional photolithographic fabrication methods. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and microRNA-155 (miR-155) were monitored in a lipopolysaccharide (LPS)-induced septic mouse model. The optimum target hybridization time in a high conductivity medium was observed to be 60 s leading to the completion of the whole operation within 5 min compared with the 4-h detection time of the traditional enzyme-linked immunosorbent assay (ELISA). The limit of detection (LOD) was calculated to be 0.84, 0.18, and 0.0014 pg mL−1, respectively. This novel sensor may have potential for the early diagnosis of sepsis in the clinical setting.

Bio-functionalized carbon dots for signaling immuno-reaction of carcinoembryonic antigen in an electrochemical biosensor for cancer biomarker detection

Early diagnosis of cancer demands sensitive and accurate detection of cancer biomarkers in blood. Carbon dots (CDs) bio-functionalization with antibodies, peptides or aptamers have played significant role in cancer diagnosis and targeted cancer therapy. Herein, a biosensor for detection of cancer biomarker carcinoembryonic antigen (CEA) in blood serum has been designed using CDs bio-functionalized with HRP-conjugated CEA antibody (CUCDs@CEAAb2) as detection probe. CDs were synthesized by upscaling of cow urine, a nitrogen rich biomass waste, by hydrothermal method. Detection probe based on CDs resulted in 3.5 times higher sensitivity as compared to conventional electrochemical sandwich immunoassay. To further improve the sensor performance, hyper-branched polyethylenimine grafted poly amino aniline (PEI-g-PAANI) was used as the sensing interface, which enabled immobilization of higher amount of capture antibody. Detection of CEA in human blood serum coupled with wide linear range (0.5–50 ng/ml), good specificity, stability, reproducibility and low detection limit (10 pg/ml) signified the excellence of CUCDs based CEA immunosensor. CUCDs exhibited excitation wavelength dependent fluorescence property and showed strong blue emission under UV irradiation. MTT assay indicated that the material is not toxic towards human dental pulp stem cells (hDPSCs) and MG63 osteosarcoma cells (cell viability > 90%). The present study demonstrates a methodology for valorization of animal waste to a cost-effective carbon based functional nanomaterial for clinical detection of cancer biomarkers.