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We propose a concept for ligase detection by conversion of aggregation-based homogeneous analysis into surface-tethered electrochemical assay through streptavidin (SA)-biotin interaction. Sortase A (SrtA) served as the model analyte and two biotinylated peptides (bio-LPETGG and GGGK-bio) were used as the substrates. SrtA-catalyzed ligation of the peptide substrates led to the generation of bio-LPETGGGK-bio. The ligation product (bio-LPETGGGK-bio) induced the aggregation and color change of SA-modified gold nanoparticles (AuNPs) through the SA-biotin interactions, which could be assayed by the colorimetric method. Furthermore, we found that the bio-LPETGGGK-bio could trigger the assembly of tetrameric SA proteins with the formation of the (SA-bio-LPETGGGK-bio)n assemblies through the same interactions. The above results were further confirmed by atomic force microscopy and fluorescent imaging. The insulated assemblies were in-situ fabricated at the SA-modified gold electrode, thus hindering the electron transfer of [Fe(CN)6]3−/4− and leading to an increase in the electron-transfer resistance. The capability of the method for the detection of SrtA both in vitro and Staphylococcus aureus (S. aureus) has been demonstrated. SrtA with a concentration down to 1 pmol/L has been determined by the electrochemical analysis, which is lower than that achieved by the colorimetric assay (50 pmol/L). By integrating the advantages of homogeneous reaction and heterogeneous detection, the strategy serves as an ideal means for the fabrication of various sensing platforms by adopting biotin-labeled and sequence-specific peptide or nucleic acid substrates.

Chemiluminescence immunoassay (CLIA) has always been a great challenge in detecting cardiac troponin I (cTnI) in whole blood samples without centrifugation because of the interference of red blood cells and low sensitivity. In this study, the antigens and erythrocytes in the blood were captured by the antibodies immobilized on the magnetic particles, recognized by another biotin-conjugated cTnI antibody and detected by streptavidin/acridine aster-conjugated polychloromethylstyrene microspheres (PCMS). After magnetic separation, the supernatant was transferred and measured. No significant difference was noted between the cTnI concentrations of the serum samples, plasma samples and whole blood. The prepared PCMS provided more functional areas to conjugate streptavidin and acridinium ester, so the immunoassay has highly sensitive, the limits of blank at 0.012 ng/mL, and functional sensitivity at 0.019 ng/mL with a CV of 20%, and 0.058 ng/mL with a CV of 10%. Total precision of any sample type ranged from 2.62%~5.67%. The assay was linear over the studied range of 0.01–50.00 ng/mL, and no hook effect was found when cTnI concentrations reached 1900 ng/mL. No significant interference was noted with the potential endogenous interfering substances. Compared with the commercial kit (Abbott assay kit), the correlation coefficient was 0.9859. A washing-free CLIA was established for the rapid detection of cTnI in human whole blood, using erythrocyte capture antibodies-conjugated magnetic nanoparticles for eliminating the influence of erythrocytes and PCMS for signal amplification, which showed great potential in clinical application.

Elevated blood biotin levels may interfere with some biotin-streptavidin immunoassays, used in clinical laboratories to aid diagnosis. The objective of this study was to determine the prevalence of elevated blood biotin levels in three at risk patient cohorts, where misclassification of disease status would have a high clinical impact. This retrospective, single-center study screened residual, de-identified plasma samples (N = 700) from adult patients undergoing routine thyroid stimulating hormone (TSH) (n = 500), procalcitonin (PCT) (n = 100), or human immunodeficiency virus (HIV) (n = 100) testing using the Elecsys® BRAHMS PCT (Roche Diagnostics), Access TSH (3rd IS) (Beckman Coulter Inc), and ARCHITECT HIV Ag/Ab Combo (Abbott Laboratories) immunoassays, respectively, for elevated levels of biotin (quantified by gas chromatography-time of flight mass spectrometry). Patients taking biotin supplements were included and dosages recorded from medical records. In the overall study cohort, blood biotin levels ranged 0.1-21.3 ng/mL; 44.3% (310/700) of samples were

Background The objective of this study was to estimate the prevalence of biotin supplementation in United States emergency department patients using a multi-site, geographically distributed sampling model. Methods Biotin was measured using an Abbott ARCHITECT Biotin research use only assay in 7118 emergency department patient serum or plasma samples from five US medical centers. Samples with biotin ≥10 ng/mL underwent additional LC-MS/MS confirmatory testing for biotin and its primary metabolites . The overall and site-specific prevalence of detectable biotin was determined using the screening assay while biotin speciation (i.e., prevalence of detectable metabolites) was determined using LC-MS/MS. Results Of 7118 samples screened, 291 (4.1%) had biotin ≥10 ng/mL and were considered positive. Across five medical centers, the fraction of positive samples ranged from 2.0% to 5.4%. The maximum biotin concentration observed was 355 ng/mL. Of the 285 positive screens that underwent additional LC-MS/MS testing, 89 (31%) showed detectable biotin, bisnorbiotin, and/or biotin sulfoxide . Biotin, bisnorbiotin, and biotinsulfoxide were detected in 82/89 (92.1%), 61/89 (68.5%), and 18/89 (20.2%) samples, respectively; biotin was detected in the absence of either metabolite in 18/89 (20.2%) samples. Conclusions Using a screening assay, 4.1% of emergency department patient samples were found to be potentially susceptible to interference from biotin. Confirmatory testing showed detectable biotin and/or biotin metabolites in 31% of positive screens (1.3% overall). The prevalence of biotin ≥10 ng/mL varied 2–3-fold across US emergency department patient cohorts. Biotin metabolites were observed in 80% of samples confirmed to have detectable biotin species by LC-MS/MS, suggesting that rigorous assessments of assay susceptibility to biotin interference, often performed using in vitro studies , should consider the potential role of biotin metabolites present in vivo.

In this work, a dual-labeled multiple aptasensor that can simultaneously detect multiple antibiotics was constructed, with kanamycin (KAN) and tobramycin (TOB) used as the model analytes. The aptasensor reported here featured three key elements, namely the RNA-based aptamer strands, semiconductor quantum dots (QDs), and gold nanoshells (AuNSs). Due to the high-affinity pairing between streptavidin (SA) and biotin (Bio), the two biotinylated aptamers (kanamycin aptamer, KAP; tobramycin aptamer, TAP) responsible for the specific recognition of KAN and TOB were conjugated to SA-coated cadmium sulfide (CdS) and lead sulfide (PbS) QDs, respectively for the syntheses of KAP-Bio-SA-CdS and TAP-Bio-SA-PdS composites. Moreover, the AuNSs had a very high loading efficiency for the as-prepared two composites on the gold electrode surface due to their outstanding surface-area-to-volume ratio. In the presence of target antibiotics, CdS and PbS QDs were released from AuNSs followed by dissolution into their respective metal ions for stripping analysis by differential pulse voltammetry (DPV). As a result, intensified signals from DPV peaks were obtained due to the large amount of the metal ion labels dissolved in the solution. The devised aptasensor exhibited wider detection linear range (KAN, 1–4 × 102 nM; TOB, 1–1 × 104 nM) and lower detection limits (KAN, 0.12 nM; TOB, 0.49 nM) compared to the previously reported sensors. Furthermore, the detection of KAN and TOB spiked in milk samples was successfully demonstrated. Therefore, the proposed aptasensor provides a novel sensitive platform for multi-antibiotics detection.

17β-Estradiol (E2), an important endocrine disrupting compound, could be quantitatively detected by fluorescence resonance energy transfer (FRET) aptasensor, designed in this paper. Metal organic frameworks have large specific surface area and easily modifiable groups, which are helpful for the construction of aptasensor. Specifically, streptavidin was immobilized on the synthesized MIL-53-NH2 by covalent bonding, and further linked with the biotin modified E2 aptamer (apt) through specific bonding between avidin and biotin to obtain the FRET donor probe (MIL-53-apt). Meanwhile, complementary DNA (cDNA) modified Ru(bpy)32+-doped silica nanoparticles (RuSiO2-cDNA) were prepared through covalent bonding. They acted as the FRET acceptor probe, since its absorption spectrum showed large overlap with the emission spectrum of MIL-53-apt. In the presence of E2, aptamer modified donor probes tended to bind with E2, owing to their higher selectivity and affinity. Therefore, the optimal distance between FRET pairs was broken, resulting in the fluorescence emission recovery of donor and the fluorescence emission of acceptor decreased. Under optimal conditions, this proposed aptasensor displayed sensitive detection of E2 ranging from 0.5 to 1000 nM with a detection limit of 0.2 nM. Furthermore, the sensor provides a promising method for rapid and sensitive detection of other small biological molecules.

DNA methylation plays an important role in a variety of human diseases. Thus, accurately analyze 5-methylcytosine in different DNA segments is of great significance. Herein, we proposed a novel 3D matrixed DNA self-nanocatalyzer via gold nanoparticles (AuNPs) supporting DNA self-hybridization with hemin as biomimetic enzyme and methylene blue (MB) as electrochemical mediator, which was employed as an efficient electrochemical sensitizer for the ultrasensitive bioassay of DNA 5-methylcytosine. Meanwhile, the AuNPs, graphitic carbon nitride (g-C3N4) and reduced graphene oxide (rGO) was prepared as AuNPs/g-C3N4@rGO nanocomposites to coat on the electrode surface to immobilize the capture hairpin DNA (CH). In the presence of target DNA with 5-methylcytosine, the target DNA could hybridize with CH via the hyperstable triple-helix formation. Based on the specific biorecognition between biotin and streptavidin and immune recognition between anti-5-methylcytosine antibodies and 5-methylcytosine sites on the target DNA, the 3D matrixed DNA self-nanocatalyzer could be captured onto the electrode surface to generate an amplified electrochemical signal related to the concentration of 5-methylcytosine. Under the optimal conditions, the proposed strategy performed a linear range from 10−17 M to 10−8 M with a detection limit of 8.6 aM. Remarkably, this strategy could be expanded easily to various biomarkers, including protein, DNA, phosphorylation and glycosylation, providing a promising strategy for clinical diagnosis and mechanism investigation of various diseases.

This study demonstrated a facile, green and bioinspired approach to synthesize protein-posnjakite nanobiohybrid with rod-assembled hollow shuttle-like structure. Through the one-pot mild coprecipitation process, the inorganic mineral posnjakite (Cu4(SO4) (OH)6·H2O) served as a nanocarrier to efficient co-immobilization of recognition protein (streptavidin) and enzyme (horseradish peroxidase) for signal amplification, which avoids tedious linking or purification procedures and significantly simplifies the synthetic process. This nanobiohybrid was then utilized as the signal tag for immunoassays and presented excellent performance for the detection of insecticidal crystalline (Cry) protein Cry1Ab, in the linear range of 0.1–40 ng mL−1, with the limit of detection of 63 pg mL−1. This proposed strategy is expected to the integration of a variety of biomolecules with posnjakite to design diverse multifunctional nanobiohybrids for multiple applications extending from biosensors, catalysis and biomedicine to environmental science and energy.

The application of inhibitors facilitates the stable preservation of enzyme in liquid detergent by mitigating the proteolytic activity of subtilisin. The conventionally used subtilisin inhibitors such as boric acid pose a threat to the environment and human health. Thus, the formulation of novel subtilisin inhibitors demands immediate attention. In the current study, we have screened the peptide inhibitors for subtilisin by employing the in vitro mRNA display technique. It is a sensitive screening technique with a high library capacity. The affinity screening was performed between the biotin-modified subtilisin immobilized on the streptavidin magnetic beads and the cDNA-mRNA-peptide fusion molecular library acquired from the in vitro translation and reverse transcription. The candidate peptides with high affinity were obtained after multiple rounds of screening. Furthermore, the inhibitory effect was evaluated, showing that some candidate peptides had inhibitory effects, but the isothermal titration calorimetry and time dependent experiments ultimately proved that these candidate peptides were not stable inhibitors. However, the in vitro mRNA display method explored in this study can be used as a preliminary screening method to provide candidate peptides for the screening of subtilisin inhibitors.

In this study, we fabricated sensors to detect Alzheimer’s disease (AD) biomarkers – amyloid beta (1-42) (Aβ1-42) based on localized surface plasmon resonance (LSPR). The sensors were consisted of ligand-exchanged gold nanoparticles (Au NPs) deposited on polyethylene terephthalate substrate using Langmuir-Blodgett (LB) technique. Monoclonal antibodies (anti-Aβ1-42) were then immobilized as a conjugate form with biotin onto the LB films of ligand-exchanged Au NPs by the aid of streptavidin. The attachment of the biomarkers to the antibodies immobilized on the LB films was detected by measuring absorbance change of plasmonic response peak. The sensor structure was optimized by comparing the results for the Au NPs LB films with different size and film thickness. The optimized sensor was used to detect biomarker at different concentrations in the buffer solution and a diluted cerebrospinal fluid (CSF) solution. As results, the sensor was able to detect even a trace amount of Aβ1-42 as small as 1 pg/ml from the CSF. This prototype sensor has a great potential because it shows several advantages of facile and inexpensive fabrication, and early detection of the AD.

As virus spread can lead to severe epidemics and pandemics associated with high mortality, it is necessary to have a highly sensitive detection method for viruses. Although various detection methods have been developed so far, current methods in detecting a virus require preprocessing and involve quite intricate processes of low sensitivity. Here, we have developed a virus detection method with a broad dynamic range and high sensitivity, based on immuno-complex formation between quantum dot (QD)-embedded silica nanoparticles (QD2) and magnetic beads. The multiple QD- containing QD2s showed 500 times stronger photoluminescence than individual QDs. When biotin was immobilized as a ligand, streptavidin was detected in a range of 10 zM to 10 nM. The clinical applicability of the QD2-based system was examined using the avian virus (i.e., H1N1 influenza virus), and it showed a detection range of 4.76 × 10−4 ∼ 3.2 hemagglutination unit/mL. This result is comparable to the polymerase chain reaction method, and is approximately 2100 times more sensitive than the conventional hemagglutination method. Since the QD2-based system could detect target molecules with high sensitivity without requiring an amplification step, it can be applied in various biomedical and clinical fields.

The biolayer interferometry (BLI), one promising biosensing technique for real-time, high-sensitive detection of analytes, is a new surface-sensitive spectroscopic technique in which the change of biolayer thickness is reflected by measuring the interferometric phase deflections. In our previous research, an ssDNA aptamer, 39nt, with definite interaction characteristics for recombinant human erythropoietin-α (EPO-α) was screened. In this work, a new aptameric BLI assay of EPO-α based on this aptamer 39nt was established. The biotinylated aptamer 39nt was firstly immobilized onto the optical fiber streptavidin biosensor chip, and the analytes in microliter volume were quickly diffused via high-speed vibration of 2200 r/min. The key parameters such as the orientation of aptamer sequence, the concentration for immobilization, and the overcome of nonspecific adsorption to exhibit the affinity recognition of aptamers were optimized. It was found that the 3'-end biotinylated aptamer 39nt with 50 nmol/L provided the best sensitivity, and the addition of Tween 20 and BSA could efficiently overcome nonspecific adsorption effect. Besides, towards the original designed disposable sensor chip, the affinity between protein EPO-α and ssDNA aptamer 39nt for ca. 10 times was still maintained under optimized regeneration condition, which reflected the operational flexibility of single-channel BLI system. Furthermore, a signal amplification approach of a sandwiched format via wheat germ agglutinin for detection of EPO-α was established, with the linear range of 10–200 nmol/L, and the LOD was 5 nmol/L. This assay was directly applied to the complex matrix such as 50% human plasma with recoveries in the range of 86.7%–104.2% and RSDs less than 15%. The BLI sensing system developed here provided a helpful way for practical applications in label-free protein interaction and detection.

Surface modification to obtain high dispersion stability and biocompatibility is a key factor for bio-application of upconversion nanoparticles (UCNPs). A systematic study of UCNPs modified with four hydrophilic molecules separately, comparing their dispersion stability in biological buffers and cellular biocompatibility is reported here. The results show that carboxyl-functionalized UCNPs (modified by 3,4-dihydrocinnamic acid (DHCA) or poly(monoacryloxyethyl phosphate (MAEP)) with negative surface charge have superior even-distribution in biological buffers compared to amino-functionalized UCNPs (modified by (aminomethyl)phosphonic (AMPA) or (3-Aminopropyl)triethoxysilane (APTES)) with positive surface charge. Subsequent investigation of cellular interactions revealed high levels of non-targeted cellular uptake of the particles modified with either of the three small molecules (AMPA, APTES, DHCA) and high levels of cytotoxicity when used at high concentrations. The particles were seen to be trapped as particle-aggregates within the cellular cytoplasm, leading to reduced cell viability and cell proliferation, along with dysregulation of the cell cycle as assessed by DNA content measurements. The dramatically reduced proportion of cells in G1 phase and the slightly increased proportion in G2 phase indicates inhibition of M phase, and the appearance of sub-G1 phase reflects cell necrosis. In contrast, MAEP-modified UCNPs are bio-friendly with increased dispersion stability in biological buffers, are non-cytotoxic, with negligible levels of non-specific cellular uptake and no effect on the cell cycle at both low and high concentrations. MAEP-modified UCNPs were further functionalized with streptavidin for intracellular microtubule imaging, and showed clear cytoskeletal structures via their upconversion luminescence. Statement of significance Upconversion nanoparticles (UCNP) are an exciting potential nanomaterial for bio-applications. Their anti-Stokes luminescence makes them especially attractive to be used as imaging probes and thermal therapeutic reagents. Surface modification is the key to achieving stable and compatible hydrophilic-UCNPs. However, the lack of criteria to assess molecular ligands used for ligand exchange of nanoparticles has hampered the development of surface modification, and further limits UCNP’s bio-application. Herein, we report a systematic comparative study of modified-UCNPs with four distinct hydrophilic molecules, assessing each particles’ colloidal stability in biological buffers and their cellular biocompatibility. The protocol established here can serve as a potential guide for the surface modification of UCNPs in bio-applications.

The use of “Spinach”-based RNA sensors for the detection of small metabolites and proteins has received growing interest in the recent years. While this approach can be used in vivo for cell sensing and in vitro for microfluidic assays, their overall utility is limited as a result of a high magnesium ion dependence. Here, we alleviate this limitation through incorporating a human tRNALys3 or an engineered viral F30 (a three-way junction RNA motif) scaffold to facilitate aptamer folding in vitro and improve the performance of selected streptavidin, tyrosine, and thrombin aptamers as exemplary cases. Furthermore, we demonstrate the use of a Broccoli aptamer scaffold in conjunction with the viral F30 to enable simultaneous sensing of two molecules in a logic gate type fashion. Our proof-of-concept results demonstrate the ability to redesign these aptamer sensors for improved brightness as well as signal stability without the need for high magnesium—both traits that can further enhance downstream screening applications.

Single-molecule methods, specifically single-molecule counting, convey high sensitivity in research applications. However, single-molecule counting experiments require specialized equipment or consumables to perform. We demonstrate the utility of using bright Streptavidin-Phycoerythrin (SA-PE) conjugates and an epifluorescence microscope, for single-molecule counting applications. In this work, we show that we can visualize single-molecules on glass surfaces, perform single-molecule diagnostic assays on magnetic microparticles, and image individual foci on cell surfaces. This approach is simple and effective for researchers interested in single-molecule counting.

On-site screening of biotoxins is of great importance for food safety. A new electrochemical-biosensing strategy was constructed for ochratoxin A (OTA) detection by direct using ready-made commercial portable-glucose-meter (PGM). Aptamer against OTA was adopted as the recognition probe and pre-immobilized onto the sensing interface. The complementary biotin-modified probe was further decorated by hybridization. Biotinylated invertase was further introduced onto the sensing system with streptavidin, which also acted as the signal amplification unit. The invertase, which was depended on the amount of OTA, produced the glucose from sucrose in the sensing solution. The glucose could be directly and conveniently measured with PGM. Quantitative analysis of OTA was achieved with a linear range from 0.5 ng/mL to 10 ng/mL and detection limit of 0.45 ng/mL. Of significance, it has been successfully applied for OTA analysis in rice with satisfied recoveries. This unique PGM-based electrochemical platform reveals prospective potential in food safety monitoring.

A label-free electrochemical aptasensor was developed for the sensitive detection of interferon-gamma (IFN-γ). To do this, a diblock dual-aptamer allosteric hairpin (DDAH) was designed, followed by conjugation with gold nanoparticles (DDAH&AuNP). The presence of target destroyed the stable hairpin structure, and then the catalytic cleavage of DNAzymes removed the IFN-γ-binding molecules, triggering the allosteric switching from inactive hairpin to active streptavidin aptamer (A-DDAH&AuNP) in homogeneous system. Moreover, streptavidin-inorganic hybrid nanoflowers decorated with graphene composites (SFG) were synthesized and used as substrates to modify glassy carbon electrodes (SFG/GCE). SFG specifically bind to the A-DDAH&AuNP to realize high-efficient readout of signals. Under the optimal conditions and by using differential pulse stripping voltammetry (DPSV), the response peak currents increases linearly with the logarithm of the IFN-γ concentration in the range between 0.1 pg mL−1 and 500 ng/mL. The detection limit is as low as 19 fg mL−1. The aptasensor also has excellent electrochemical performances, which exhibits broad application prospects in biometric analysis.

N-glycans are involved in a variety of biological processes and associated with many diseases. However, the sensitive and specific detection of glycans via mass spectrometry (MS) remains challenging due to their relatively low abundance and low ionization efficiency. In this work, a new method termed glycan reductive amino acid coded affinity tagging (GRACAT) is developed to address these problems. Through labeling the reducing ends of glycan with a biotinylated arginine, the ionization efficiency of glycan was increased nearly 50-fold and glycan isomers were discriminated by improved signals of fragments in tandem spectra. Moreover, the strong affinity interaction between streptavidin and biotin enabled highly specific enrichment of the biotin-tagged glycans from the mixture of proteins and peptides. We successfully applied GRACAT in the profiling of N-glycome in human serum and bovine milk, in which a total of 55 and 67 N-glycans were detected respectively.

A novel homogeneous assay system QTR-FRET (Quencher modulated Time-Resolved Forster Resonance Energy Transfer) combining quenching resonance energy transfer (QRET) and time-resolved Forster resonance energy transfer (TR-FRET) was developed to reduce background signal in the conventional energy transfer applications. The TR-FRET functionality is often limited by the lanthanide donor background signal leading to the use of low donor concentration. QTR-FRET reduces this background by introducing soluble quencher molecule, and in this work the concept functionality was proven and compared to previously introduced QRET and TR-FRET technologies. Comparison was performed with three different Eu3+-chelates exhibiting different luminescent lifetime and stability. The side-by-side comparison of the three signaling systems and Eu3+-chelates was demonstrated in a model assay with Eu3+-chelate conjugated biotin and streptavidin (SA) or Cy5-SA conjugate. Comparison of the methodologies showed increased signal-to-background ratios when comparing QTR-FRET to TR-FRET, especially at high Eu3+-biotin concentrations. Quenching the non-bound Eu3+-biotin improved the assay performance, which suggests that an improved assay performance can be attained with the QTR-FRET method. QTR-FRET is expected to be especially useful for Eu3+-labeled ligands with low affinity or assays requiring high Eu3+-ligand concentration. The QTR-FRET indicated potential for multi-analyte approaches separately utilizing the direct QRET-type Eu3+-chelate signal and energy transfer signal readout in a single-well. This potential was hypothesized with Avi-KRAS nucleotide exchange assay as a second biologically relevant model system.

Magnetic separation has been widely exploited for capture and detection of nucleic acids, including amplicons. Streptavidin-magnetic beads (SA-MB) are typically employed for this purpose, as well as in biosensing applications. However, remaining biotinylated primer in the amplification reaction can compete with labeled amplicon for binding to the beads. Also, the harsh conditions needed for elution of bound amplicons restrict their use for purification purposes. Herein we show that a sequence-specific DNA binding protein immobilized on magnetic beads can serve as an alternative to SA-MB for these applications. This is enabled by the high binding affinity of scCro DNA binding protein for its specific sequence and its ability to bind dsDNA but not ssDNA. This specific sequence is easily incorporated in the amplicon during amplification with an extended primer. The scCro-MB exhibited higher amplicon binding capacity and detection sensitivity compared to SA-MB when both synthetic and genomic DNA were used as templates for PCR. This resulted not only from increased protein load on the beads but also from minimized interference of excess labeled primer remaining in the unpurified amplification reactions. Finally, a proof-of-concept was provided for the use of the scCro-MB for PCR amplicon purification under mild elution conditions using salt.

As a crucial medium of intercellular communication in a variety of pathological process, small extracellular vesicles (sEVs) are currently expected to be a novel diagnostic biomarker and therapeutic target. However, highly sensitive and quantitative detection of sEVs remains challenging. Herein, we propose a novel sEVs sensing system through integration of CD63 aptamer based identification and exonuclease III (Exo-III) assisted signal recycling. In this method, streptavidin magnetic beads (SMBs)-Capture probe is designed to specially recognize sEVs, in which process the blocker sequences is released from the complex to induce the following signal amplification. Based on the Exo-III enzyme assisted triple signal amplification, the method exhibits a wide detection range from 10

Quantified inflammatory biomarkers are effective clinical strategy for correct and reasonable drug treatment. In the study, a triple lateral flow immunoassay (triple LFIA) had firstly been developed for specific and simultaneous detection of three pivotal inflammatory biomarkers (PCT, CRP and SAA) via biotin-streptavidin-phycoerythrin signal amplification system in one strip. The developed triple LFIA adopted phycoerythrin (PE) as chromophore to eliminate auto-fluorescence interference from plasma biomolecules and anti-PE mAb as single control line to reduce the nonspecific adsorption, which featured particular advantages in high sensitivity and specificity in a large range of analyte concentrations with the LODs of 0.106 ng/mL for PCT, 0.345 μg/mL for CRP and 3.112 μg/mL SAA, respectively. And the linear quantitative detection ranges were from 0.106 to 100 ng/mL, from 0.345 to 200 μg/mL, and from 3.112 to 200 μg/mL, respectively. Compared to commercial chemiluminescence immunoassay method, the correlations for tested PCT, CRP and SAA in 108 clinical samples were 0.989, 0.987 and 0.988, respectively. In summary, we had proposed a rapid and accurate plasma detection to measure inflammation factors, which facilitated the clinical value to achieve precise treatment.

Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His

Magnetic beads (MBs) are often considered as an effective carrier in heterogeneous assays due to the simplicity of separation and washing, and the ability to increase and control the surface area. However, the effect of the MBs surface on the analytical parameters is poorly characterized and is often postulated from intuitive considerations. Herein, experimental evaluation through the comparison of MBs and microwell plate was carried out using the miRNA-141 (biomarker for cancer) as a target, the detection of which was performed by chemiluminescent assay with a homogeneous mismatched catalytic hairpin assembly (mCHA) reaction. The mCHA reaction produced double-stranded (ds) DNA labeled at one end with fluorescein (Flu) for capture with anti-Flu antibodies immobilized on a solid carrier, on the other end with biotin for recognition by streptavidin-polyperoxidase conjugate. The conditions of immobilization of anti-Flu antibody on MBs (a diameter of 440 nm) performed using a carbodiimide method were optimized by varying the antibody concentration in the reaction solution. It was shown that the dependence of chemiluminescent signal as a function of the concentration of anti-FluAb-MBs conjugates had a bell-shaped character. The maximum chemiluminescence was produced at the concentration of the conjugates of 2 × 10

Prodrugs that present strong cytotoxicity toward specific cells have been utilized for cell-type selection and purification. In this study, we designed and prepared a multifunctional, photoactivated prodrug based on a streptavidin scaffold. Biotin-labeled DNA aptamer that recognizes the membrane antigen EpCAM, and biotin-labeled photoactivated prodrug bearing the antitumor camptothecin, were prepared. Both molecules were linked to the streptavidin scaffold by simple mixing. The resulting prodrug bound to the EpCAM-overexpressing SK-BR-3 target cells and showed cytotoxic effects upon photoirradiation, corresponding to cytotoxic drug release.

It is very meaningful and useful to select specific aptamers with capacity to distinguish small structural analogues, but it is difficult to carry out by traditional affinity chromatography-SELEX (systematic evolution of ligands by exponential enrichment) based on immobilized target molecules. In this paper, as a proof of concept, we selected DNA aptamers that can specifically recognize and differentiate riboflavin and its derivative flavin adenine dinucleotide (FAD) by a modified method. Here, the random DNA library was indirectly immobilized on streptavidin functional agarose beads by hybridization with its biotinylated short complementary strand, and the specific affinity between aptamers and its target would induce the aptamers to release from beads. Binding specificity can be tailored by performing an additional negative SELEX with the structure analogue of target. After about 10 rounds of selection, 6 aptamers for riboflavin and 2 aptamers for FAD with good affinities were isolated, and their dissociation constants (Kds) were all at low micromolar level. Moreover, as expected, most of these aptamers show high affinity and excellent selectivity for target molecules, almost no binding to structure analogues and purines, indicating this simple method could be used to select specific aptamers to distinguish small molecular targets with similar structures.

Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest (for example, at its primary amines) and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, whether this method can be used for samples where interaction with the air-water interface has been shown to be problematic remains to be determined. Here we report a 3.1 A structure of an RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)’s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids improved the angular distribution of its view, resulting in a high-resolution structure. This work shows that streptavidin affinity grids can be used to address known challenges posed by the interaction with the air-water interface.

Biosensor surface plasmon resonance (SPR) is a highly sensitive technique and is most commonly used to decipher the interactions of biological systems including proteins and nucleic acids. Throughout the years, there have been significant efforts to develop SPR assays for studying protein-protein interactions, protein-DNA interactions, as well as small molecules to target DNAs that are of therapeutic interest. With the explosion of discovery of new RNA structures and functions, it is time to review the applications of SPR to RNA interaction studies, which have actually extended over a long time period. The primary advantage of SPR is its ability to measure affinities and kinetics in real time, along with being a label-free technique and utilizing relatively small quantities of materials. Recently, developments that use SPR to analyze the interactions of different RNA sequences with proteins and small molecules demonstrate the versatility of SPR as a powerful method in the analysis of the structure-function relationships, not only for biological macromolecules but also for potential drug candidates. This chapter will guide the reader through some background material followed by an extensive assay development to dissect the interactions of small molecules and RNA sequences using SPR as the critical method. The protocol includes (i) fundamental concepts of SPR, (ii) experimental design and execution, (iii) the immobilization of RNA using the streptavidin-biotin capturing method, and (iv) affinities and kinetics analyses of the interactions using specific example samples. The chapter also contains useful notes to address situations that might arise during the process. This assay demonstrates SPR as a valuable quantitative method used in the search for potential therapeutic agents that selectively target RNA.

Biotinylated antibodies/antigens are currently used in many immunoassay formats in clinical settings for diversified analytes and biomarkers to offer high detection selectivity and sensitivity. Biotin cannot be synthesized by mammals and must be taken as an essential supplement. Normal intake of biotin from various foods and milk causes no effect on the streptavidin/biotin-based immunoassays. However, overconsumption of biotin (daily doses 100–300 mg) poses a significant problem for immunoassays using the biotin-strept(avidin) pair. Biotin interferences are noted in immunoassays of thyroid markers, drugs, hormones, cancer markers, the biomarker for cardiac function (β–human chorionic gonadotropin), etc. The biotin level required for serious interference in test results varies significantly from test to test and cannot easily be predicted. Immunoassay manufacturers with technologies based on strept(avidin)-biotin binding must investigate the interference from biotin (up to at least 1200 ng/mL or 4.9 μM of biotin) in various formats. There is no concrete solution to circumvent the biotin interference encountered in blood samples, short of biotin removal. Considering the short half-life of biotin in the human body, patients must stop taking biotin supplements for >48 h before the test. However, this scenario is not considered for patients in emergency situations or those with biotinidase deficiency, mitochondrial metabolic disorders or multiple sclerosis. Apparently, a rapid analytical procedure for biotin is urgently needed to quantify for its interference in immunoassays using strep(avidin)-biotin chemistry. To date, there is no quick and reliable procedure for the detection of biotin at below nanomolar levels in blood and biological samples. Traditional lab-based techniques including HPLC/MS-MS cannot process an enormous number of public samples. Biosensors with high detection sensitivity, miniaturization, low cost, and multiplexing have the potential to address this issue.

Human papillomavirus (HPV) is a double-standard DNA virus, as well as the source of infection to the mucous membrane. It is a sexually transmitted disease that brings the changes in the cervix cells. Oncogenes, E6 and E7 play a pivotal role in the HPV infection. Identifying these genes to detect HPV strains, especially a prevalent HPV16 strain, will bring a great impact. Among different sensing strategies for pathogens, the dielectric electrochemical biosensor shows the potential due to its higher sensitivity. In this research, HPV16-E7 DNA sequence was detected on the carbodiimidazole-modified interdigitated electrode (IDE) surface with the detection limit of 1 fM. To enhance the sensitivity, the target sequence was conjugated on gold nanoparticle (GNP) and attained detection to the level of 10 aM. This produced ~100 folds improvement in detecting HPV16-E7 gene and 4 folds increment in the current flow. The stability of HPV16-E7 DNA sequences on GNP was verified by the salt-induced GNP aggregation. The current system has shown the higher specificity by comparing against non-complementary and triple-mismatched DNA sequences of HPV16-E7. This demonstration in detecting HPV16-E7 using dielectric IDE sensing system with a higher sensitivity can be recommended for detecting a wide range of disease-causing DNA-markers.

Two high-affinity DNA aptamers for lung tumor cells were applied as biospecific elements in bioluminescent assay of patient blood. The oligonucleotide complementary to the 5′ end of both aptamers carrying either biotin or Ca2+-regulated photoprotein obelin was used to form a sandwich-type analytical complex on the surfaces of magnetic streptavidin-activated microspherical particles. Clinical blood samples from cases of morphologically confirmed lung cancer and control samples were analyzed applying the developed assay. From the receiver operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers the sensitivity of 91.5% and the specificity of 75% (p

A DNA-based bead array method was successfully developed to simultaneously discriminate 11 pathogens namely Listeria grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, L. welshimeri, Escherichia coli, E. coli O157:H7, Staphylococcus aureus, methicillin-resistant S. aureus, and Salmonella spp. for multi purposes: food safety, hygiene indication, antibiotic resistance treatment. The bead array technology is based on fluorescent-barcoded paramagnetic beads with unique 24 oligonucleotide (anti-TAG) sequences which can capture biotinylated PCR product with complementary TAG sequence. R-phycoerythrin labeled streptavidin is used to report the presence of the biotinylated PCR products. After optimizing assay conditions, amplification and biotinylation steps can be performed in a single reaction without further purification before hybridization between the biotinylated TAG products and anti-TAG beads. To ensure that the developed method could provide accurate testing with the real food sample, a total of 311 bacterial isolates from 194 chicken meat samples were tested. The results were compared with those from the conventional ISO methods and revealed the relative accuracy, relative specificity, and relative sensitivity of 96%, 100%, and 95%, respectively. Therefore, the developed method was demonstrated to be useful to distinguish 11 bacteria species at the same time with high accuracy, specificity, and sensitivity.

Background Red blood cells (RBCs) can be labeled with N-hydroxysuccinimidobiotin (sulfo-NHS-biotin), which binds to cell surface proteins under aqueous conditions. Biotinylated RBCs can be safely infused and detected in peripheral blood samples using flow cytometry, using a fluorochrome-conjugated streptavidin (SA) detection reagent. Biotinylated RBCs have been used to track survival of transfused RBCs, and have applications in optimizing RBC storage and in understanding donor genetic, environmental and disease factors affecting RBC products. Methods We have developed a closed-system, current good manufacturing practices (cGMP)–compliant procedure for biotinylation of RBCs and a quantitative flow cytometric assay to estimate the dose of cell-bound biotin delivered to the patient. Resulting products were characterized for variability, sterility, endotoxin, hemolysis, total dose of cell-bound biotin and stability. Results The density of biotin-labeling increased as a log-linear function of sulfo-NHS-biotin–labeling concentration, with greater variability at lower concentrations. The upper estimates of biotin doses in the average product (mean RBC content = 5.55 × 1011) were 9.8 and 73.0 µg for products labeled at 3 and 15 µg sulfo-NHS-biotin/mL of total reaction mixture (27 and 135 nmol/mL packed RBCs), respectively. All products were negative for bacterial and fungal growth at 14 days and were below the limit of endotoxin detection. Biotinylated RBCs were stable in vitro for up to 50 days after labeling. Discussion We have validated a closed-system procedure for biotinylating RBCs for investigational use. A standard operating procedure is presented in sufficient detail for implementation in a cGMP-compliant cell-processing facility.

Encapsulation of guest molecules into the vacant space of biomacromolecular crystals has been utilized for various purposes including functioning as a protein container to protect against physical stress and structural determination of the guest. Todarodes pacificus hemocyanin (TpHc) is a hollow cylindrical decameric protein complex with an inner space 110 A in diameter and 160 A in height. In the crystal, TpHc forms a straw-like bundle and contains one reactive Cys (Cys3246) in the inner domain of each protomer. Here, we conjugated biotin onto Cys3246 of TpHc followed by incubation with streptavidin. The streptavidin was immobilized into the inner space of TpHc due to its interaction with biotin. Moreover, the complex containing TpHc and streptavidin was crystallized under the same conditions used for unmodified TpHc. In order to expand this methodology for a variety of proteins, we conjugated the ligand nitrilotriacetic acid (NTA) chelated to a Ni2+ ion (Ni2+-NTA) to TpHc. We found that His-tagged green fluorescent protein (GFP) was encapsulated into the Ni2+-NTA-conjugated TpHc via the interaction between the His-tag and the Ni2+-NTA group. X-ray crystallography demonstrated that the crystal packing of the complex containing TpHc and GFP was identical to that of the unmodified TpHc. Our guest immobilization method is distinct from previous approaches that are dependent on diffusion of the guest into the host crystal. Thus, our findings may accelerate the development of proteinaceous crystal engineering.

This paper presents a technology computer-aided design (TCAD) analysis of an ultrasensitive In2O5Sn gate (transparent gate) recessed channel (TGRC) metal-oxide-semiconductor field effect transistor (MOSFET) as a biosensor for early-stage disease diagnostics. The key parameters such as sensitivity, switching ratio, and threshold voltage shift have been compared with the conventional MOSFET. For immobilizing the protein molecules, a cavity has been embedded in the gate insulator region due to which gate capacitance changes owing to the accumulation of protein molecules which reflects the deviation in threshold voltage. Higher sensitivity (1.542) is achieved for protein at a very low drain bias (0.2 V) in comparison to streptavidin and APTES ((3-Aminopropyl) triethoxysilane). Moreover, the cavity gap variation (from 8 to 15 nm) and oxide thickness limitation has also been observed for the device as a biosensor. All the results pave way for early detection techniques of protein-related diseases such as Alzheimer's diseases, ovarian cancer and coronary artery disease with the existing complementary metal oxide semiconductor (CMOS) technology.

Abnormal expression of microRNAs is closely related to human diseases. Ultrasensitive detection of microRNAs with wide linear dynamic range is necessary as the concentrations of microRNAs distributed in biological samples usually range from fM to nM. Here, we constructed an ultrasensitive electrochemical sensor for microRNA-21 based on the integration of a dual signal amplification strategy of hybridization chain reaction (HCR) and enzyme-induced metallization (EIM) with anodic stripping voltammetry (ASV). MicroRNA-21 was captured by capture probe H1 (CP H1) modified magnetic nanobeads (MBs) and amplified by HCR. Multiple alkaline phosphatases (ALP) were coupled based on the specific reaction of biotin and streptavidin. A dual amplification strategy of HCR and EIM enhanced electrochemical signal by approximately 120-fold from the perspectives of target amplification and detection signal amplification, which decreased the detection limit of microRNA-21 to 0.12 fM. Compared to the reports previously reported, a wider linear dynamic range of 2.5 fM to 25 nM spanning 7 orders of magnitude was obtained. The method provides high sensitivity, specificity and a wider linear dynamic range, which shows a great potential in the early diagnosis of diseases.

In this work, we developed and optimized conjugates of carbon-coated iron nanoparticles (Fe@C) with streptavidin and monoclonal antibodies. The conjugation procedure included two stages. First, amino groups were grafted onto the carbon shell to facilitate noncovalent sorption of bovine serum albumin (BSA). Further, the covalent attachment of proteins to the BSA layer via glutaraldehyde coupling was performed. It was established and confirmed that the synthesis procedure is reproducible and allows preparation of stable conjugates. The resulting nanoparticles are clusters of Fe@C particles coated by proteins. The size of the clusters is in the range of 100–190 nm and can be controlled via the tuning of conjugation conditions, including pH, BSA-to-Fe@C ratio, etc. Conjugates of Fe@C with streptavidin and monoclonal antibodies (sizes of approximately 140–150 nm) were synthesized. Proton T2 relaxometry was used to detect these conjugates with very high sensitivity due to the magnetic markers, Fe@C. The relaxivity (r2) of different conjugates varied within the range of 290–450 1/s*mM. Conjugate applicability for relaxometry-based assay was confirmed by direct detection of streptococcal protein G and biotinylated BSA in a dot immunoassay.

Controlling foodborne pathogens is important to ensure food safety and prevent foodborne diseases. In this study, a Fe3O4 nanoparticle cluster (Fe3O4 NPC) biosensor based on nuclear magnetic resonance was developed for Salmonella detection. The capture antibody of Salmonella was used, and the biotinylated antibody specifically recognised Salmonella at different sites. Fe3O4 NPC modified with streptavidin were used to capture biotinylated antibody by biotin and determine streptavidin interaction. Finally, Fe3O4 NPC immobilised on 96-well microplates were eluted, thereby reducing the transverse relaxation time (T2) of neighbouring water molecules. The method detected Salmonella at 105 cfu mL−1, which was also the limit of detection in spiked milk samples, and showed high selectivity over other non-target bacteria. The proposed biosensor could be a potential tool for sensitive and rapid detection of foodborne pathogens in food, environmental, and agricultural samples.

This article is clearly presenting the development of a biosensor for human factor IX (FIX) to diagnose the blood clotting deficiency, a so-called ‘Royal disease’ using an interdigitated electrode (IDE) with the zinc oxide surface modification. Gold nano-urchins (GNUs) with 60 nm in diameter was integrated into a streptavidin-biotinylated aptamer strategy to enhance the active surface area. Two different comparative studies have been done to validate the system to be practiced in the current work holds with a higher capability for the high-performance sense. Whereby, the presence and absence of GNUs in the aptasensing system for FIX interaction were investigated using the amperometric measurement, using a linear sweep voltage of 0–2 V at 0.01 V step voltage. The detection limit was 6 pM based on 3σ calculation when GNUs integrated aptamer assay was utilized for FIX detection, which shows 8 folds sensitivity enhancement comparing the condition in the absence of GNU and 50 folds higher than sensitive radio-isotope and surface plasmon resonance assays. Albeit, the surface and molecular characterizations were well demonstrated by scanning electron microscopy, atomic force microscopy, 3D nano-profilometry and further supports were rendered by UV–Vis spectroscopy and Enzyme-linked apta-sorbent assay (ELASA). Furthermore, the spiking experiment was done by FIX-spikes in human blood serum in order to demonstrate the stability with a higher non-fouling.

Human papillomavirus (HPV) type 16 is widely distributed in population, which is responsible for more than 50% of cervical cancers. A point-of-care testing for HPV16 is critical for early diagnosis and decisions regarding patient care. In this work, a novel lateral flow strip biosensor (LFSB) has been established by a simple, low-cost and green synthetic procedure for the detection of HPV16 DNA. Copper oxide nanoparticles (CuO NPs) were used as colored tags in the LFSB for the first time. In a typical experimental procedure, CuO NPs were sequentially functionalized with streptavidin and biotinylated capture DNA to form the CuO Capture probes. In the present of HPV16 DNA, the CuO Capture probes would capture the target, forming a characteristic brown band due to the second base pairing with the test DNA probes on the test zone of LFSB, which enabling the visual detection. A portable strips reader achieved the quantitative detection. The optimized CuO-based LFSB was capable of detecting the target over its concentration ranged from 5 nM to 100 nM with a limit of detection of 1.0 nM, and the whole detection process could be completed within 20 min. The CuO-based LFSB shows a great promise and versatile opportunities in point-of-care diagnosis of genetic diseases with good reproducibility and stability.

A biotin-streptavidin-amplified enzyme-linked immunosorbent assay using a biotinylated nanobody (BA-Nb ELISA) was developed to detect ochratoxin A (OTA) in cereal. The limit of detection (LOD) of the BA-Nb ELISA, which equals to 10% maximal inhibitory concentration, was 0.011 ng/mL for OTA in buffer, and the sensitivity was approximately improved by one order of magnitude compared with the traditional Nb ELISA (LOD = 0.112 ng/mL). Under optimal conditions, the developed assay could be accomplished in 40 min with maximal inhibitory concentration of 0.138 ng/mL and the linear detection range of 0.034–0.460 ng/mL. The average recovery rate of the BA-Nb ELISA ranged from 92.8% to 114%, and the relative standard deviation was in the range of 2.04–9.85%. The developed BA-Nb ELISA was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the results indicated the reliability of BA-Nb ELISA for the detection of OTA in cereal.

Diffusion sensors for nano-sized materials dispersed in a solution are promising tools for investigating intermolecular interactions, which is an important task in a wide range of industrial and biomedical fields, such as the development of therapeutic products and diagnostic tools. For the development of the sensor applicable to point-of-care testing, an optofluidic sensor was proposed. The proposed sensor realizes a simple measurement in a short time using microscale manipulation by interferometrically-induced dielectrophoresis and optical detection. In this paper, the applicability of the proposed sensor to distinguish nanoscale differences in size was confirmed by measurements on a solution containing size-certified plain nano-beads (51 nm, 100 nm, 203 nm, 216 nm and 240 nm) with evaluating the uncertainties of the proposed sensor. Furthermore, measurements using 200-nm beads coated with functional groups (NH2 and biotin) and proteins (streptavidin and bovine serum albumin) demonstrated the applicability to the investigation of intermolecular interactions.

While the binding of biotin by streptavidin does not appear to be cooperative in the traditional sense of altered binding strength, it has been suggested that it may be cooperative in terms of differential structural changes in the protein. In this work we present intrinsic tryptophan fluorescence data as evidence of a cooperative structural change. The technique involves examination of the differences in fluorescence emission corresponding to distinct tryptophan populations accompanying protein-ligand binding. Specifically we note that the 335 nm emission population (i.e. more hydrophobic) saturates prior to the saturation of the 350 nm emission population commonly used in the standard binding activity assay. We also note that the wavelength of maximum emission, total integrated fluorescence emission and full width at half maximum during the titration of ligand into streptavidin also reach saturation before the expected 4:1 stoichiometric end point. This suggests that the binding of the first 3 biotins effect greater structural changes in the protein than the final ligand., Highlights • Structurally sensitive intrinsic Trp fluorescence changes upon biotin addition. • Key features of this fluorescence stops changing prior to full biotin saturation. • This suggests that ligand-induced structural changes are allosteric. • We introduce use of ‘cooperative allosterism’ to distinguish from traditional usage.

Quantifying eggs from Haemonchus and other trichostrongyle genera in sheep and goat fecal samples is important for evaluating control and treatment strategies for this family of nematodes with divergent pathologies, capabilities for anthelmintic resistance and environmental susceptibilities. Unfortunately, egg morphology among most of the genera do not differ enough to support the accurate identification of these genera with standard microscopic techniques. Several studies have identified specific lectins which bind selectively to sugars located on the egg surfaces for individual genera among the trichostrongyles. To detect lectins binding to these eggs, they must be directly or indirectly bound to fluorophores, and observed with an epi-fluorescence microscope. The binding of multiple lectins to isolated eggs from a fecal sample can be simultaneously detected if fluorophores are used whose excitation and emission spectra do not overlap, and this would enable the development of a fluorescence-based diagnostic test that identifies multiple trichostrongyle genera within each sample. The present study compared the usefulness of different, commercially available detection systems for use in detecting lectin binding to trichostrongyle eggs. Comparisons were made using the detection of PNA binding to H. contortus eggs with the goal of finding three systems with color spectra that do not overlap. These evaluations included both fluorophores directly conjugated to PNA in a one-step incubation protocol and a two-step incubation protocol involving biotinylated PNA and streptavidin conjugated to different fluorophores. Autofluorescence can affect the efficiency of any fluorescence-based detection system, and significant autofluorescence was observed among the unstained H. contortus eggs with the DAPI-type fluorescence filter, but it was significantly lower with the FITC-type filter and was virtually absent with the rhodamine-type filter. This study demonstrated that all the PNA detection methods tested with H. contortus eggs generated fluorescence intensities (FIs) that were significantly above the autofluorescence generated by the eggs among the three different fluorescence filters. Fluorescence intensities from PNA directly conjugated to either the FITC or rhodamine fluorophores were not different, but the lower autofluoresence in the rhodamine-type filter will enable this fluorophore to be detected more efficiently. Use of biotinylated PNA combined with streptavidin-conjugated to synthetic fluorophores (Alexa Fluor 405, 488 and 546) significantly increased FIs over that of the directly conjugated PNA, but there were no significant differences in FIs among these three biotin-avidin conjugation fluorophores. This biotin-avidin system required two incubation steps. Doubling the concentration of PNA also provided increased FI, at least for the biotin-avidin system. Adding an additional amplification step to the biotin-avidin system involving biotinylated anti-streptavidin followed by the streptavidin-Alexa Fluor complex also provided additional fluorescence.

A miniaturized optical immunosensor based on White Light Reflectance Spectroscopy (WLRS) for the rapid and label-free detection of aflatoxin M1 (AFM1) in milk samples has been developed. WLRS sensing system consists of the measurement set-up and the biochip. The first encompasses the reflection probe, the light source and the spectrometer, while the latter is a Si chip with a SiO2 layer on top where an AFM1-bovine serum albumin conjugate has been immobilized. The assay was performed by running mixtures of rabbit polyclonal anti-AFM1 antibody with the calibrators or the samples, followed by reaction with biotinylated anti-rabbit IgG antibody and streptavidin. The assay cycle was completed in 25 min, the limit of detection was 6 pg/mL, and the linear working range extended from 0.012 to 2.0 ng/mL. The assay was repeatable (intra-and inter-assay coefficients of variation ranged from 2.1 to 6.3% and 3.5 to 8.2%, respectively) and accurate (percent recovery values ranged from 92.5 to 110%). AFM1 could be detected with the immunosensor developed in both processed and unprocessed milk of different animal species without any dilution. The excellent analytical characteristics and the small instrument size make the proposed sensor suitable for accurate low-cost AFM1 determination in milk samples at the point-of-need.

This work proposes a dielectric modulated GaAs junctionless FinFET as a biological sensor in the sub-20 regime. In the proposed biosensor, HfO2 (ᴋ = 25) is used as a base oxide which is found to have improved the switching ratio (three times) of the device. For immobilizing the biomolecules, a Nano-cavity (18 nm) is embedded between gate and source/drain. For the detection of biomolecules, electrical characteristics such as switching ratio, energy band profile and change in surface potential have been studied and thereafter, sensitivity has been evaluated which has been improved by 80%. Change in current is recorded for different materials due to change in gate capacitance owing to different biomolecules: Protein (ᴋ = 8), streptavidin (ᴋ = 2.1), biotin (ᴋ = 2.63). Higher sensitivity is observed for protein biomolecules (1.07) as compared to streptavidin (1.015) and biotin (1.045). The sensitivity is compared to with the absence of biomolecules (assumed as the vacuum) as well. Furthermore, modulation of the cavity gap length was also investigated, revealing that its increase (from 8 to 18 nm) significantly enhanced the sensitivity of the proposed biosensor. All the results pave way for biomolecule detection with the existing CMOS technology.

A simple and selective photoelectrochemical method was reported for 5-hydroxymethylcytosine (5hmC) detection in genomic DNA based on DNA methyltransferase (MTase) catalytic covalent bonding of –CH2OH of 5hmC with thiol compounds, where WS2 nanomaterial was used as a photoactive material, polydopamine (PDA) was employed as a solid-phase electron donor, phos-tag-biotin was used as bridge to link 5hmC and streptavidin. PDA was attached to 4-mercaptophenulboronic acid (MPBA) via the covalent bonding between its vicinal diol and the boracic acid of MPBA. Then, under the catalytic effect of M. HhaI MTase, 5hmC deoxyribonucleotide was captured on electrode surface through the covalent bonding between –CH2OH and –SH. Afterwards, phos-tag-biotin, a kind of specific reagent for phosphate group, was further captured through the specific bonding between the phosphate group of 5hmC deoxyribonucleotide and phos-tag-biotin. Subsequently, based on the “bridge” role of phos-tag-biotin, streptavidin was further modified on electrode surface to further block the transfer of photogenerated-electron to electrode surface. The photocurrent shows linear relationship with the logarithm value of 5hmC concentration in the range from 0.01 to 100 nM, and the detection limit of 4.12 pM (3σ). The developed method presents high detection specificity, which can discriminate 5-methylcytosine and 5hmC. Moreover, the applicability of this photoelectrochemical method was evaluated by detecting 5hmC content change in maize seedlings leaves and chicken embryo fibroblast cell.

Introducing clickable groups onto polymer microspheres allows the microspheres to be facilely functionalized via a click reaction. In this report, we have synthesized monodisperse PMMA microspheres with clickable double bonds on the surface by one-step RAFT-assisted dispersion photopolymerization using a cyclohexyl-containing macro-RAFT agent as a stabilizer. This macro-RAFT agent, named P(THPAA-b-AA)-TTC, was synthesized by RAFT-mediated block copolymerization of a cyclohexyl-containing monomer (THPAA, obtained by esterification of cis-1,2,3,6-tetrahydrophthalic with 2-hydroxyethyl acrylate) and acrylic acid. The obtained microspheres were analyzed by 1H NMR and Iodometric method, indicating that the content of carbon double bonds on the microspheres increased with increasing the amount of P(THPAA-b-AA)-TTC in the polymerization feed. We then used a thiolated biotin to perform the photo-click reaction with the cyclohexene double bond on the microspheres, thereby attaching the biotin unit onto the microspheres. The biotinylated PMMA microspheres were found to be able to effectively immobilize fluorescein isothiocyanate streptavidin (FITC-SAv) via the specific adsorption of biotin to streptavidin, and thus exhibited clear fluorescence under confocal microscope. This study provides a convenient and feasible way to prepare surface-functionalized microspheres, which has great potential in biological applications as well as other fields.

Antibody-mediated rejection (AMR) has emerged as a major cause of renal allograft dysfunction. C4d, a specific marker for AMR diagnosis, was strongly recommended for routine surveillance; however, currently, C4d detection is dependent upon tissue biopsy, which is invasive and provides only local semi-quantitative data. Targeted ultrasound imaging has been used extensively for noninvasive and real-time molecular detection with advantages of high specificity and sensitivity. In this study, we designed C4d-targeted microbubbles (MBC4d ) using a streptavidin-biotin conjugated method and detected C4d deposition in vivo in a rat model of AMR by enhanced ultrasound imaging. This noninvasive procedure allowed successful acquisition of the first qualitative image of C4d deposition in a wide renal allograft section, which reflected real-time C4d distribution in grafts. Moreover, we introduced normal intensity difference for quantitative analysis, which exhibited a nearly linear correlation with the grade of C4d deposition according to pathologic analysis. In addition, this approach showed no influence on survival rates and pathologic features in the microbubble injection groups, thereby demonstrating its safety. These findings demonstrated a simple, noninvasive, quantitative, and safe evaluation method for C4d, with the utility of this approach potentially preventing patients from having to undergo an invasive biopsy.