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Accurate quantification of neurofilament lights (NfLs), a prognostic blood biomarker, is highly required to predict neurodegeneration in the presymptomatic stages of Alzheimer's disease. Here, we report self-oxygen-enriching coral structures with triphase interfaces for the label-free photocathodic detection of NfLs in blood plasma with femtomolar sensitivities and high reliability. In conventional photocathodic immunoassays, the poor solubility and sluggish diffusion rate of the dissolved oxygen serving as electron acceptors have necessitated the incorporation of additional electron acceptors or aeration procedures. To address the challenge, we designed the coral-like copper bismuth oxides (CBO) with robust solid-liquid-air contact boundaries that enrich the interfacial oxygen levels without an external aeration source. By optimally assembling the perfluorododecyltrichlorosilane (FTCS) and platinum (Pt) co-catalysts into the silver-doped CBO (Ag:CBO), the stable solid-liquid-air contact boundaries were formed within the sensor interfaces, which allowed for the abundant supply of air phase oxygen through an air pocket connected to the atmosphere. The Pt/FTCS-Ag:CBO exhibited the stable background signals independent of the dissolved oxygen fluctuations and amplified photocurrent signals by 1.76-fold, which were attributed to the elevated interfacial oxygen levels and 11.15 times-lowered mass transport resistance. Under the illumination of white light-emitting diode, the oxygen-enriching photocathodic sensor composed of Pt/FTCS-Ag:CBO conjugated with NfLs-specific antibodies precisely quantified the NfLs in plasma with a low coefficient of variation (≤2.97%), a high degree of recovery (>97.0%), and a limit of detection of 40.38 fg/mL, which was 140 times lower than the typical photocathodic sensor with diphase interfaces.

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Pre-eclampsia (PE) is a life-threatening complication that occurs during pregnancy, affecting a large number of pregnant women and newborns worldwide. Rapid, on-site and affordable screening of PE at an early stage is necessary to ensure timely treatment and minimize both maternal and neonatal morbidity and mortality rates. Placental growth factor (PlGF) is an angiogenic blood biomarker used for PE diagnosis. Herein, we report the plasmonic fiber optic absorbance biosensor (P-FAB) strategy for detecting PlGF at femtomolar concentration using polymethyl methacrylate (PMMA) based U-bent polymeric optical fiber (POF) sensor probes. A novel poly(amidoamine) (PAMAM) dendrimer based PMMA surface modification is established to obtain a greater immobilization of the bioreceptors compared to a linear molecule like hexamethylenediamine (HMDA). Plasmonic sandwich immunoassay was realized by immobilizing the mouse anti-PlGF (3H1) on the U-bent POF sensor probe surface and gold nanoparticles (AuNP) labels conjugated with mouse anti-PlGF (6H9). The POF sensor probes could measure PlGF within 30 min using the P-FAB strategy. The limit-of-detection (LoD) was found to be 0.19 pg/mL and 0.57 pg/mL in phosphate-buffered saline and 10× diluted serum, respectively. The clinical sample testing, with eleven positive and eleven negative preeclamptic pregnancy samples, successfully confirmed the accuracy, reliability, specificity, and sensitivity of the P-FAB based POF sensor platform, thereby paving the way for cost-effective technology for PlGF detection and its potential for pre-eclampsia diagnosis.

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In addressing the demand for hierarchically mesoporous metal-organic frameworks (HMMOFs) with adjustable large mesopores, a method based on the synergistic effects of low-temperature microemulsions and Hofmeister ions is developed. Low temperature dramatically enhanced the solubility of hydrophobic solvent in the microemulsion core, enlarging the mesopores in HMMOFs replica. Meanwhile, Hofmeister salt-in ions continuously controlled mesopore expansion by modulating the permeability of swelling agent into the microemulsion core. The large mesopores up to 33 nm provided sufficient space for the alkaline phosphatase (ALP) enrichment, and retained the remaining channel to facilitate the free mass diffusion. Leveraging these advantages, a colorimetric sensor is successfully developed using large-mesopore HMMOFs for femtomolar ALP detection based on the enrichment and cycling amplification principles. The sensor exhibited a linear detection range of 100 to 7500 fm and a limit of detection of 42 fm, presenting over 4000 times higher sensitivity than classic para-nitrophenyl phosphate colorimetric methods. Such high sensitivity highlights the importance of adjustable mesoporous structures of HMMOFs in advanced sensing applications, and prefigures their potential for detecting large biomolecules in diagnostics and biomedical research.

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Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.

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The histone methyltransferase EZH2 has been the target of numerous small-molecule inhibitor discovery efforts over the last 10+ years. Emerging clinical data have provided early evidence for single agent activity with acceptable safety profiles for first-generation inhibitors. We have developed kinetic methodologies for studying EZH2-inhibitor-binding kinetics that have allowed us to identify a unique structural modification that results in significant increases in the drug-target residence times of all EZH2 inhibitor scaffolds we have studied. The unexpected residence time enhancement bestowed by this modification has enabled us to create a series of second-generation EZH2 inhibitors with sub-pM binding affinities. We provide both biophysical evidence validating this sub-pM potency and biological evidence demonstrating the utility and relevance of such high-affinity interactions with EZH2.

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Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, accompanies a massive degradation of neurons including axonal injury. Being an axonal neuron-specific protein, neurofilament light (NfL) is a blood biomarker that reflects the neurodegeneration in AD, but no attempt has been made yet to develop sensing platforms that target NfLs in blood serum or plasma. Here, we report three-dimensional cross-stacked Pt nanowire arrays for the ultrasensitive photoelectrochemical (PEC) detection of NfLs. We constructed a woodpile-like Pt nanowire array (PtWP)-based biocathode by printing multilayer Pt nanowire arrays in an orthogonal configuration and conjugating them with NfL-specific DA2 antibodies. According to our collective electrochemical analyses, the five-layered PtWP electrode modified with DA2 antibodies exhibited high oxygen reduction activities due to the large electrochemical active surface area and the effective electron transfer properties. We have combined the DA2-PtWP biocathode with a water-oxidizing, iron oxyhydroxide-deposited bismuth vanadate (FeOOH/BiVO4) photoanode to assemble a bias-free PEC detection system. Powered by a white-light-emitting diode, the unbiased PEC platform accurately recognizes NfLs in blood plasma with the limit-of-detection of 38.2 fg/mL and limit-of-quantification of 853 fg/mL, which is 40 times lower than the NfL levels in AD patients' blood.

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The detection of chemical messenger molecules, such as neurotransmitters in nervous systems, demands high sensitivity to measure small variations, selectivity to eliminate interferences from analogues, and compliant devices to be minimally invasive to soft tissue. Here, an organic electrochemical transistor (OECT) embedded in a flexible polyimide substrate is utilized as transducer to realize a highly sensitive dopamine aptasensor. A split aptamer is tethered to a gold gate electrode and the analyte binding can be detected optionally either via an amperometric or a potentiometric transducer principle. The amperometric sensor can detect dopamine with a limit of detection of 1 µM, while the novel flexible OECT-based biosensor exhibits an ultralow detection limit down to the concentration of 0.5 fM, which is lower than all previously reported electrochemical sensors for dopamine detection. The low detection limit can be attributed to the intrinsic amplification properties of OECTs. Furthermore, a significant response to dopamine inputs among interfering analogues hallmarks the selective detection capabilities of this sensor. The high sensitivity and selectivity, as well as the flexible properties of the OECT-based aptasensor, are promising features for their integration in neuronal probes for the in vitro or in vivo detection of neurochemical signals.

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Accurate detection of target molecules at low concentrations in the presence of undesired molecules in abundance is a major challenge for biosensors. Nonspecific binding of undesired molecules to receptors limits the minimum detectable concentration of the target significantly. Dynamic tracking (DT) of binding and unbinding events allows us to overcome this challenge and provides a remarkable improvement in the minimum detectable target concentration. Through a combination of theoretical analysis and detailed statistical simulations, here we show that, with aggressive scaling, DT sensors are capable of fM detection limits even if the undesired molecules are present at nM concentrations, which is several orders of magnitude better than traditional endpoint (EP) biosensors. In addition, we propose a novel unconstrained detection scheme that does not rely on a priori knowledge of the dissociation constants and also allows facile back-extraction of critical parameters. Indeed, this work provides a theoretical basis for DT sensors and demonstrates its suitability to usher in a new paradigm on biosensing in hostile environments.

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Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. A key pathogenic event of AD is the formation of intracellular neurofibrillary tangles that are mainly composed of tau proteins. Here, we report on ultrasensitive detection of total tau (t-tau) proteins using an artificial electron donor-free, BiVO4-based photoelectrochemical (PEC) analysis. The platform was constructed by incorporating molybdenum (Mo) dopant and iron oxyhydroxide (FeOOH) ad-layer into the BiVO4 photoelectrode and employing a signal amplifier formed by horseradish peroxidase (HRP)-triggered oxidation of 3,3'-diaminobenzidine (DAB). Despite the absence of additional electron suppliers, the FeOOH/Mo:BiVO4 conjugated with the Tau5 antibody produced strong current signals at 0 V (vs. Ag/AgCl, 3 M NaCl) under the illumination of a white light-emitting diode. The Mo extrinsic dopants increased the charge carrier density of BiVO4-Tau5 by 1.57 times, and the FeOOH co-catalyst promoted the interfacial water oxidation reaction of Mo:BiVO4-Tau5 by suppressing charge recombination. The introduction of HRP-labeled Tau46 capture antibodies to the FeOOH/Mo:BiVO4-Tau5 platform produced insoluble precipitation on the transducer by accelerating the oxidation of DAB, which amplified the photocurrent signal of FeOOH/Mo:BiVO4-Tau5 by 2.07-fold. Consequently, the water oxidation-coupled, FeOOH/Mo:BiVO4-based PEC sensing platform accurately and selectively recognized t-tau proteins down to femtomolar concentrations; the limit of detection and limit of quantification were determined to be 1.59 fM and 4.11 fM, respectively.

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Quantitation of biomarkers in biofluids plays a central role in basic research to management of patient care and is routinely used in clinical laboratories and academic institutions. Standard immunoassays, such as an enzyme-linked immunosorbent assay (ELISA), have provided understanding of both normal and pathological processes for many decades. However, in more recent decades, new immunoassay technologies have uncovered numerous analytes in blood that were once undetectable using traditional ELISAs. To meet this new challenge for quantifying low abundant proteins in biofluids, Single Molecule Counting (SMC™) technology was developed. This new technology is a combination of improvements to both the immunoassay procedure as well as the instrument. The aim of this article is to introduce the new SMCxPRO™ instrument, xPRO Acquisition and Analysis software, and the high sensitivity immunoassay kits validated on this instrument for the detection of low abundant proteins in biofluids, such as serum and plasma. Using this new technology platform, biomarkers that were once unquantifiable can now be quantitated in both normal and diseased biofluids.

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The advent of optical fiber-based biosensors combined with that of nanotechnologies has provided an opportunity for developing in situ, portable, lightweight, versatile, and high-performance optical sensing platforms. We report on the generation of lossy mode resonances by the deposition of nanometer-thick metal oxide films on optical fibers, which makes it possible to measure precisely and accurately the changes in optical properties of the fiber-surrounding medium with very high sensitivity compared to other technology platforms, such as long period gratings or surface plasmon resonances, the gold standard in label-free and real-time biomolecular interaction analysis. This property, combined with the application of specialty structures such as D-shaped fibers, permits enhancing the light-matter interaction. SEM and TEM imaging together with X-EDS tool have been utilized to characterize the two films used, i.e., indium tin oxide and tin dioxide. Moreover, the experimental transmission spectra obtained after the deposition of the nanocoatings have been numerically corroborated by means of wave propagation methods. With the use of a conventional wavelength interrogation system and ad hoc developed microfluidics, the shift of the lossy mode resonance can be reliably recorded in response to very low analyte concentrations. Repeated experiments confirm a big leap in performance thanks to the capability to detect femtomolar concentrations in human serum, improving the detection limit by 3 orders of magnitude when compared with other fiber-based configurations. The biosensor has been regenerated several times by injecting sodium dodecyl sulfate, which proves the capability of sensor to be reused.

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Numerous proteases, such as matrix metalloproteinases (MMPs), cathepsins (CTS), and urokinase plasminogen activator (UpA), are dysfunctional (that is, over- or under-expressed) in solid tumors, when compared to healthy human subjects. This offers the opportunity to detect early tumors by liquid biopsies. This approach is of particular advantage for the early detection of pancreatic cancer, which is a "silent killer". We have developed fluorescence nanobiosensors for ultrasensitive (sub-femtomolar) arginase and protease detection, consisting of water-dispersible Fe/Fe3O4 core/shell nanoparticles and two tethered fluorescent dyes: TCPP (Tetrakis(4-carboxyphenyl)porphyrin) and cyanine 5.5. Upon posttranslational modification or enzymatic cleavage, the fluorescence of TCPP increases, which enables the detection of proteases at sub-femtomolar activities utilizing conventional plate readers. We have identified an enzymatic signature for the detection of pancreatic adenocarcinomas in serum, consisting of arginase, matrix metalloproteinase-1, -3, and - 9, cathepsin-B and -E, urokinase plasminogen activator, and neutrophil elastase, which is a potential game-changer.

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This paper presents the development of tapered optical fiber sensor to detect a specific Leptospira bacteria DNA. The bacteria causes Leptospirosis, a deadly disease but with common early flu-like symptoms. Optical single mode fiber (SMF) of 125 µm diameter is tapered to produce 12 µm waist diameter and 15 cm length. The novel DNA-based optical fiber sensor is functionalized by incubating the tapered region with sodium hydroxide (NaOH), (3-Aminopropyl) triethoxysilane and glutaraldehyde. Probe DNA is immobilized onto the tapered region and subsequently hybridized by its complementary DNA (cDNA). The transmission spectra of the DNA-based optical fiber sensor are measured in the 1500 to 1600 nm wavelength range. It is discovered that the shift of the wavelength in the SMF sensor is linearly proportional with the increase in the cDNA concentrations from 0.1 to 1.0 nM. The sensitivity of the sensor toward DNA is measured to be 1.2862 nm/nM and able to detect as low as 0.1 fM. The sensor indicates high specificity when only minimal shift is detected for non-cDNA testing. The developed sensor is able to distinguish between actual DNA of Leptospira serovars (Canicola and Copenhageni) against Clostridium difficile (control sample) at very low (femtomolar) target concentrations.

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Altered serum and plasma microRNA (miRNA) expression profiles have been observed in numerous human diseases, with a number of studies describing circulating miRNA biomarkers for cancer diagnosis, prognosis and response to treatment, and recruitment to clinical trials for miRNA-based drug therapy already underway. Electrochemical detection of biomarkers in urine has several significant advantages over circulating biomarker analysis including safety, cost, speed and ease of conversion to the point of care environment. Consequently, much current research is underway to identify urinary miRNA biomarkers for a variety of pathologies including prostate and bladder malignancies, and renal disorders. We describe here a robust method capable of electrochemical detection of human urinary miRNAs at femtomolar concentrations using a complementary DNA-modified glassy carbon electrode. A miR-21-specific DNA hybridisation probe was immobilised onto a glassy carbon electrode modified by sulfonic acid deposition and subsequent chlorination. In our pilot system, the presence of synthetic mature miR-21 oligonucleotides increased resistance at the probe surface to electron transfer from the ferricyanide/ferrocyanide electrolyte. Response was linear for 10 nM-10 fM miR-21, with a limit of detection of 20 fM, and detection discriminated between miR-21, three point-mutated miR-21 sequences, and miR-16. We then demonstrated similar sensitivity and reproducibility of miR-21 detection in urine samples from 5 human control subjects. Our protocol provides a platform for future high-throughput screening of miRNA biomarkers in liquid biopsies.

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In this study, we designed and fabricated an electrochemical impedance aptasensor based on Au nanoparticles (Au-NPs) coated boron-doped diamond (BDD) modified with aptamers, and 6-mercapto-1-hexanol (MCH) for the detection of bisphenol A (BPA). The constructed BPA aptasensor exhibits good linearity from 1.0×10-14 to 1.0×10-9molL-1. The detection limitation of 7.2×10-15molL-1 was achieved, which can be attributed to the synergistic effect of combining BDD with Au-NPs, aptamers, and MCH. The examine results of BPA traces in Tris-HCl buffer and in milk, UV spectra of aptamer/BPA and interference test revealed that the novel aptasensors are of high sensitivity, specificity, stability and repeatability, which could be promising in practical applications.

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Several nanomaterials and techniques for the detection of mercuric ions (Hg(2+)) have been developed in the past decade. However, simple, low-cost and rapid sensor for the detection of heavy metal ions yet remains an important task. Herein, we present a highly sensitive electrochemical sensor for the femtomolar detection of Hg(2+) based on polypyrrole, pectin, and graphene (PPy/Pct/GR) which was prepared by one step electrochemical potentiodyanamic method. The effect of concentration of pectin, polypyrrole and graphene were studied for the detection of Hg(2+). The influence of experimental parameters including effect of pH, accumulation time and accumulation potential were also studied. Different pulse anodic stripping voltammetry was chosen to detect Hg(2+) at PPy/Pct/GR/GCE modified electrode. The fabricated sensor achieved an excellent performance towards Hg(2+) detection such as higher sensitivity of 28.64µAµM(-1) and very low detection limit (LOD) of 4 fM at the signal to noise ratio of 3. The LOD of our sensor offered nearly 6 orders of magnitude lower than that of recommended concentration of Hg(2+) in drinking water by United States Environmental Protection Agency and World Health Organization. Compared to all previously reported electrochemical sensors towards Hg(2+) detection, our newly fabricated sensor attained a very LOD in the detection of Hg(2+). The practicality of our proposed sensor for the detection of Hg(2+) was successfully demonstrated in untreated tap water.

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Proteases, including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins (CTS) exhibit numerous functions in tumor biology. Solid tumors are characterized by changes in protease expression levels by tumor and surrounding tissue. Therefore, monitoring protease levels in tissue samples and liquid biopsies is a vital strategy for early cancer detection. Water-dispersable Fe/Fe3O4-core/shell based nanoplatforms for protease detection are capable of detecting protease activity down to sub-femtomolar limits of detection. They feature one dye (tetrakis(carboxyphenyl)porphyrin (TCPP)) that is tethered to the central nanoparticle by means of a protease-cleavable consensus sequence and a second dye (Cy 5.5) that is directly linked. Based on the protease activities of urokinase plasminogen activator (uPA), MMPs 1, 2, 3, 7, 9, and 13, as well as CTS B and L, human breast cancer can be detected at stage I by means of a simple serum test. By monitoring CTS B and L stage 0 detection may be achieved. This initial study, comprised of 46 breast cancer patients and 20 apparently healthy human subjects, demonstrates the feasibility of protease-activity-based liquid biopsies for early cancer diagnosis.

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A sequence-specific oligonucleotide detection method based on the tail-to-tail aggregation of functionalized gold nanoparticles in the presence of target analytes is presented together with its optimization and capabilities for detection of single nucleotide polymorphisms (SNPs). In this single-step method, capture probes are freely accessible for hybridization, resulting in an improved assay performance compared to substrate-based assays. The analytes bring the nanoparticles close to each other via hybridization, causing a red shift of the nanoparticle plasmon peak detected by a spectrophotometer or CCD camera coupled to a darkfield imaging system. Optimal conditions for the assay were found to be (i) use of capture probes complementary to the target without any gap, (ii) maximum possible probe density on the gold nanoparticles, and (iii) 1M ionic strength buffer. The optimized assay has a 1 fM limit of detection and fM to 10 pM dynamic range, with detection of perfect match sequences being three orders of magnitude more sensitive than targets with single nucleotide mismatches.

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In this paper we demonstrate an efficient and non-interfering computational method for sub-femtomolar food toxin detection in complex mixture based on nanoporous silicon oxide impedance immunosensor by employing noise spectroscopy analysis at the peak frequency. It has been observed that the peak frequency (fp) values obtained from steady state impedance measurements cannot distinguish between solution with only the specific toxin, which is aflatoxin B1 (AfB1) and mixture of AfB1 with other non-specific toxins (NSTs), thus leading to erroneous quantification of AfB1 in complex mixture. On the other hand, the first cut-off frequency (fc) ranges obtained from noise spectroscopy analysis can qualitatively differentiate between solution containing only AfB1, AfB1 and NSTs and no AfB1. However fc values being very close for different concentration of AfB1 in pure solution and being overlapping for different mixtures cannot quantify AfB1 either in pure solution or in complex mixture. To address this problem, the proposed computational method first clusters the fp and fc values in 11 categories each using k-means clustering algorithm and then applies a simple combinational digital logic on the clusters of fps and fcs to obtain the final output, realizable with standard NAND-NOR gates. The output digital word differs only with AfB1 concentration and not with concentration of NSTs and is found to be capable of detecting sub-femtomolar AfB1 range down to 0.1 fg/ml not only in pure solution but also in complex mixture with as high as 1000 ng/ml NSTs. This is the most sensitive and selective report so far on electrochemical food toxin immunosensors.

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To determine the role of proteins, and in particular protein variants, in human health, it may often be necessary to quantitatively determine the concentration of a specific protein variant present in complex biological samples such as blood, cerebral spinal fluid (CSF), or tissue. Many protein variants are present only at trace levels and therefore a simple assay with very high sensitivity and reliability would greatly facilitate correlation of the presence of particular protein variants with the progression of specific diseases. We have developed a simple phage based capture ELISA system that enables femtomolar or better detection of individual protein variants directly from complex biological samples. The protocol utilizes a capture reagent that selectively recognizes a unique epitope of the protein variant and a phage based detection reagent that binds to a second epitope present in all forms of the target protein. The phage based detection reagent is essentially a self-assembling nanoparticle consisting of several thousand coat proteins that can each be labeled to amplify the detection signal by several orders of magnitude. Here we demonstrate that we can achieve subfemtomolar detection of individual protein variants that have been implicated in neurodegenerative disease directly from complex tissue homogenates and sera. The ELISA system should facilitate identification of disease specific protein variants or other compounds even when present at trace amounts in samples including blood, CSF, saliva and urine.

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