Biotin-streptavidin Binding Research Articles

Controlling the Magnetic Responsiveness of Cellulose Nanofiber Particles Embedded with Iron Oxide Nanoparticles.

2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanofiber (TOCN) particles, an innovative biobased material derived from wood biomass, have garnered significant interest, particularly in the biomedical field, for their distinctive properties as biocompatible particle adsorbents. However, their microscopic size complicates their separation in liquid media, thereby impeding their application in various domains. In this study, superparamagnetic magnetite nanoparticles (NPs), specifically iron oxide Fe3O4 NPs with an average size of 15 nm, were used to enhance the collection efficiency of TOCN-Fe3O4 composite particles synthesized through spray drying. These composite particles exhibited a remarkable ζ-potential (approximately -50 mV), indicating their high stability in water, as well as impressive magnetization properties (up to 47 emu/g), and rapid magnetic responsiveness within 60 s in water (3 wt % Fe3O4 to TOCN, 1 T magnet). Furthermore, the influence of Fe3O4 NP concentrations on the measurement of the speed of magnetic separation was quantitatively discussed. Additionally, the binding affinity of the synthesized particles for proteins was assessed on a streptavidin-biotin binding system, offering crucial insights into their binding capabilities with specific proteins and underscoring their significant potential as functionalized biomedical materials.

A rapid magneto-optical immunoassay method based on liquid optical cavities

The technique investigates the application of the AC magneto-optical dichroism of magnetic fluids in fast and low-cost immunoassays by taking advantage of liquid optical cavities. Magnetic nanoparticles (MNPs) served as biomarkers. Variations in particle size were characterized by monitoring the amplitude of the second harmonic of transmitted light under alternating magnetic field excitation. Differential beams with polarization directions parallel and perpendicular to the magnetic field can improve signal-to-noise ratio. We designed a liquid optical cavity that allowed multiple reflections of light in magnetic fluid to increase the optical path. This cavity enhanced the magneto-optical signal and lowered the detection limits of target molecules. The limit of detecting biotin in the streptavidin–biotin binding system in conjunction with MNPs was 0.027 μg/mL and the least amount of time required for detection was 2.3 s. It is expected to achieve a low-cost, compact, and portable biosensor for rapid detection of trace viruses.

Molecular beacon-peptide probe based double recycling amplification for multiplexed detection of serum exosomal microRNAs.

Exosomal microRNAs (exomiRs) have been shown to play crucial roles as biomarkers for early detection and prognosis of cancer. However, simultaneous quantification of multiplex exomiRs is hindered by methods that require additional steps, such as labeling with fluorophores or gel visualization, which are susceptible to various factors. Herein, we developed a mass spectrometry-detectable and target-triggered method for multiplexed exomiR detection using three enzyme-based double recycling amplification in combination with well-designed molecular beacon-peptide (MBP) probes, called molecular beacon-peptide probe-based double recycling amplification (MBPDRA). MBP probes mediated the double recycling amplification reaction and were released as mass-detectable reporter peptides. In particular, the hybridization of the target microRNAs (miRNAs) with the stem-loop of the probe triggers two consecutive processes. The first cycle involved polymerase strand displacement amplification, leading to the production of complementary DNA (cycle I), and the second cycle encompassed the recycling exonuclease cleavage of the MBP probe (cycle II). Subsequently, excess probes were removed by interaction with streptavidin beads via biotin-streptavidin binding. The reporter peptides were released using trypsin and subsequently detected by mass spectrometry. Our method enables quantitative detection of multiple exomiRs with a dynamic range from 0.1 fM to 10 pM and a limit of quantification of 0.1 fM. Moreover, the proposed assay was successfully employed for quantification of three exomiRs, exmiR-21, exmiR-191, and exmiR-451a, in the sera of patients with pancreatic cancer. Based on these findings, we believe that the MBPDRA assay holds significant promise as a reliable method for quantifying multiple miRNAs in biomedical research and clinical diagnostics.

Electrochemiluminescence sensing of HeLa cells labeled with biotinylated ruthenium complex using bipolar electrode based on microwell modified optical fiber.

Biotinylated ruthenium complexes exhibit improved photoluminescent (PL) properties when they bind with streptavidin, making them useful labels or probes in bio-related analysis. However, their ECL properties are still unknown to date. Herein, we reported the use of [Ru(bpy)2(biot-bpy)]2+ complexes as a new ECL luminophore, which was functionalized with biotin moiety and exhibited higher ECL efficiency after binding to streptavidin. Moreover, [Ru(bpy)2(biot-bpy)]2+ complexes could be attached to HeLa cells through the biotin-streptavidin binding. A microwell bipolar electrode (MBE) prepared at one end of an optical fiber bundle was applied to produce ECL of the labeled HeLa cells, which was remotely detected at the other end. The [Ru(bpy)2(biot-bpy)]2+-streptavidin binding effect together with the high surface/volume ratio of MBE promoted the ECL generation on HeLa cells, which was applied to sensitively detect HeLa cells with a linear range from 1.56 × 102 to 6.74 × 106 cells/mL and a detection limit of 83 cells/mL. Moreover, ECL images were successfully acquired to resolve the emission on each HeLa cell. Such cytosensor based on [Ru(bpy)2(biot-bpy)]2+ and MBE may extend the applications of ECL for cell detections.

Effects of High-Biotin Sample Interference on Antibody Concentrations in Sandwich Immunoassays.

The use of antimicrobial growth promoters (AGPs) is banned because of problems associated with drug residues in animal products and increased bacterial resistance. The immunization of chickens with specific antigens is a promising strategy for generating specific antibodies that can target a wide range of antibiotic-resistant bacteria and can be used as an alternative to antibiotics. Immunoglobulin Y (IgY) antibodies in a polyclonal antibody (pAb) format, when administered orally, modulate the ruminal microbiome and maintain animal health and performance; however, there are concerns pertaining to protein impurities and biotin concentrations in the samples. Signal amplification strategies involving the noncovalent interaction of biotin with streptavidin is extensively used in diagnosis and scientific research, particularly in enzyme-linked immunosorbent assays (ELISAs). However, the high concentrations of biotin in samples, especially in those derived from rich sources such as egg yolk, can pose challenges and potentially harm the accuracy of diagnostic tests and protein concentration measurements. This study aimed to evaluate the influence of biotin on the measurement of IgY in freeze-dried egg yolk samples obtained from immunized laying hens using immunoassays with biotin-avidin/streptavidin. The detection of IgY in yolk samples using ELISA with streptavidin-biotin binding could lead to misdiagnosis due to biotin interference; the level of interference varies with the specific assay conditions and the concentration of biotin in the yolk samples. An ELISA without streptavidin-biotin binding is advisable to avoid interactions between biotin and target proteins, prevent biotin interference with the results, and achieve more reliable and accurate results.

Spatially Resolved Protein Binding Kinetics Analysis in Microfluidic Photonic Crystal Sensors

Organ-on-a-Chip systems are emerging as an important in vitro analysis method for drug screening and medical research. For continuous biomolecular monitoring of the cell culture response, label-free detection within the microfluidic system or in the drainage tube is promising. We study photonic crystal slabs integrated with a microfluidic chip as an optical transducer for label-free biomarker detection with a non-contact readout of binding kinetics. This work analyzes the capability of same-channel reference for protein binding measurements by using a spectrometer and 1D spatially resolved data evaluation with a spatial resolution of 1.2 μm. A cross-correlation-based data-analysis procedure is implemented. First, an ethanol–water dilution series is used to obtain the limit of detection (LOD). The median of all row LODs is (2.3±0.4)×10−4 RIU with 10 s exposure time per image and (1.3±0.24)×10−4 RIU with 30 s exposure time. Next, we used a streptavidin–biotin binding process as a test system for binding kinetics. Time series of optical spectra were recorded while constantly injecting streptavidin in DPBS at concentrations of 1.6 nM, 3.3 nM, 16.6 nM and 33.3 nM into one channel half as well as the whole channel. The results show that localized binding within a microfluidic channel is achieved under laminar flow. Furthermore, binding kinetics are fading out at the microfluidic channel edge due to the velocity profile.

Impact of Biotin Supplementation on Human Chorionic Gonadotropin Immunoassays Utilizing Biotin-Streptavidin Binding Methods in Urine.

Human chorionic gonadotropin (hCG) detection is indicative of pregnancy and can be indicative of some forms of cancerous tumors. The hCG drug itself, however, is a performance enhancing substance used by male athletes to increase testosterone production. Antidoping testing for hCG is conducted in urine, often on immunoanalyzer platforms, many of which utilize biotin-streptavidin dependent immunoassays in which the presence of biotin in samples is a known confounding factor. While biotin interference in serum has been well-studied, the extent of biotin interference in urine has not. Ten active male individuals underwent a 2-week hCG administration protocol concurrent with supplementation with biotin (20 mg/day) or placebo. Urine and serum samples were collected throughout the study and analyzed for hCG and biotin concentrations. Urinary biotin levels in the hCG + biotin group increased 500-fold over baseline and 29-fold over corresponding serum biotin levels after biotin supplementation. When using a biotin-dependent immunoassay, the hCG + placebo group produced hCG-positive results (hCG ≥ 5 mIU/mL) in 71% of urine samples, while the hCG + biotin group produced positive results in only 19% of samples. Both groups had elevated hCG values in serum measurements by a biotin-dependent immunoassay and in urine when using a biotin-independent immunoassay. Urinary hCG measurements and biotin levels from the hCG + biotin group showed a negative correlation (Spearman r = -0.46, P < 0.0001) when measured using a biotin-dependent immunoassay. Biotin supplementation can severely suppress urinary hCG values in assays utilizing biotin-streptavidin binding methods and therefore these types of assays are not recommended for use in urine samples containing high levels of biotin. Clinicaltrials.gov Registration Number: NCT05450900.

Entry of microparticles into giant lipid vesicles by optical tweezers.

Entry of micro- or nanosized objects into cells or vesicles made of lipid membranes occurs in many processes such as entry of viruses into host cells, microplastics pollution, drug delivery, or biomedical imaging. Here we investigate the microparticle crossing of lipid membranes in giant unilamellar vesicles in the absence of strong binding interactions (e.g., streptavidin-biotin binding). In these conditions, we observe that organic and inorganic particles can always penetrate inside the vesicles provided an external piconewton force is applied and for relatively low membrane tensions. In the limit of vanishing adhesion, we identify the role of the membrane area reservoir and show that a force minimum exists when the particle size is comparable to the bendocapillary length.

Honokiol acts as an AMPK complex agonist therapeutic in non-alcoholic fatty liver disease and metabolic syndrome

BackgroundNon-alcoholic fatty liver (NAFLD) and its related metabolic syndrome have become major threats to human health, but there is still a need for effective and safe drugs to treat these conditions. Here we aimed to identify potential drug candidates for NAFLD and the underlying molecular mechanisms.MethodsA drug repositioning strategy was used to screen an FDA-approved drug library with approximately 3000 compounds in an in vitro hepatocyte model of lipid accumulation, with honokiol identified as an effective anti-NAFLD candidate. We systematically examined the therapeutic effect of honokiol in NAFLD and metabolic syndrome in multiple in vitro and in vivo models. Transcriptomic examination and biotin-streptavidin binding assays were used to explore the underlying molecular mechanisms, confirmed by rescue experiments.ResultsHonokiol significantly inhibited metabolic syndrome and NAFLD progression as evidenced by improved hepatic steatosis, liver fibrosis, adipose inflammation, and insulin resistance. Mechanistically, the beneficial effects of honokiol were largely through AMPK activation. Rather than acting on the classical upstream regulators of AMPK, honokiol directly bound to the AMPKγ1 subunit to robustly activate AMPK signaling. Mutation of honokiol-binding sites of AMPKγ1 largely abolished the protective capacity of honokiol against NAFLD.ConclusionThese findings clearly demonstrate the beneficial effects of honokiol in multiple models and reveal a previously unappreciated signaling mechanism of honokiol in NAFLD and metabolic syndrome. This study also provides new insights into metabolic disease treatment by targeting AMPKγ1 subunit-mediated signaling activation.

Distance-controlled surface-enhanced Raman spectroscopy of nanoparticles.

Biological particles, e.g., viruses, lipid particles, and extracellular vesicles, are attracting significant research interest due to their role in biological processes and potential in practical applications, such as vaccines, diagnostics, and therapies. Their surface and interior contain many different molecules including lipids, nucleic acids, proteins, and carbohydrates. In this Letter, we show how distance-controlled surface-enhanced Raman spectroscopy (SERS) is a promising method to extract essential information from the spatial origin of the signal. This is a highly important parameter in the analysis of these biological particles. The principle of the method is demonstrated by using polystyrene (PS) beads as a biological particle model conjugated with gold nanospheres (AuNSs) functioning as distance-controlled SERS probes via biotin-streptavidin binding. By tuning the size of AuNSs, the Raman signal from the PS beads can be weakened while the signal from the biotin-streptavidin complex is enhanced.

Magnetic-bead-based DNA-capture-assisted real-time polymerase chain reaction and recombinase polymerase amplification for the detection of African swine fever virus.

African swine fever (ASF) is a deadly disease in swine caused by African swine fever virus (ASFV). The global spread of ASFV has resulted in significant economic losses worldwide. Improved early detection has been the most important first line of defense for preventing ASF outbreaks and for activating control measures. Despite the availability of rapid amplification methods, nucleic acid extraction from specimens still needs to be performed in a laboratory. To facilitate this step, we exploited the strong affinity of biotin-streptavidin binding by functionalizing streptavidin-coated magnetic beads with biotinylated oligonucleotide capture probes to efficiently capture genotype II ASFV DNA directly from crude clinical samples. The captured DNA is suitable for detection using real-time quantitative PCR (qPCR) and recombinase polymerase amplification (RPA). In this study, ASFV DNA was efficiently captured from swine feces, serum, and tissue samples. Both DNA-capture-assisted qPCR and RPA-based detection methods have a limit of detection (LOD) of 102 copies/µl, which is comparable to those of commercially available kits. In addition, an RPA-SYBR Green I method was developed for the immediate visual detection of ASFV DNA, which is time-saving and efficient for resource-limited field settings. In summary, a rapid, versatile, sequence-specific DNA capture method was developed to efficiently capture ASFV DNA from swine clinical samples and subsequent detection by qPCR and RPA, which has the potential to be used for robust screening and surveillance of ASFV and in point-of-care (POC) diagnostics.

PMON224 Newly Observed Interference of Oral Biotin with the Free Testosterone Assay? In a Woman with Postmenopausal Virilization.

Abstract Introduction Approximately 15-20% of US adults take supraphysiologic doses of biotin-containing supplements for common hair and skin problems. When consumed in high doses, competitive and non-competitive immunoassays which use the streptavidin-biotin binding complex can skew results to be either falsely high or falsely low. Case Presentation A 74 y/o Caucasian female who initially presented to her primary care physician concerned for alopecia with frontal balding, acne and moderate hirsutism. Her total testosterone was elevated with 146 ng/dL (7.0–40 ng/dL) with a perplexing normal free testosterone of 4.2 pg/mL (&amp;lt;4.2 pg/mL) in the context of a normal SHBG of 48 nmol/L (14–73 nmol/L). Androstenedione was elevated with 161 ng/dL (17–99 ng/dL) with a normal CMP, prolactin, DHEA-s and 17-hydroxyprogesterone. Transvaginal US was normal. The size of the ovaries was consistent with menopause. Abdominal and pelvic MRI showed normal adrenal glands and ovaries. She was taking between 1 to 3mg of biotin per day. Biotin was discontinued. Repeat testing revealed an elevated free testosterone of 12.7 pg/mL (0.2 - 3.7 pg/mL) along with an elevated total testosterone of 120 ng/dL (2 - 45 ng/dL) and a normal SHBG of 39 nmol/L (14–73 nmol/L) which was now consistent with her clinical picture of postmenopausal hyperandrogenism. Bilateral laparoscopic salpingo-oophorectomy was recommended to exclude an androgen-secreting ovarian tumor or hyperthecosis. Testing was then repeated while taking biotin which again manifested a normal free testosterone of 2.8 pg/mL (&amp;lt;4.2 pg/mL) with an elevated total testosterone of 121.4 ng/dL (7.0–40 ng/dL) and a normal SHBG of 45 nmol/L (14–73 nmol/L). Discussion Biotin may interfere with the total testosterone levels, but little is known of its effects on free testosterone. We report the first case of biotin interference on the free testosterone assay, resulting in falsely low values demonstrated in two different occasions, that improved following withdrawal of this widely used vitamin supplement. Laboratories use a direct analog enzyme immunoassay to calculate the free testosterone however its calibration and specificity has been debatable, and it is still unclear how biotin can affect it. It is critical that laboratorians and clinicians are aware of this biotin interference so that misdiagnosis and inappropriate treatment can be prevented.

Intracellular Protein Photoactivation Using Sterically Bulky Caging.

Methods for intracellular protein photoactivation have been studied to elucidate the spatial and temporal roles of proteins of interest. In this study, an intracellular protein photoactivation method was developed using sterically bulky caging. The protein of interest was modified with biotin via a photocleavable linker, and then conjugated with streptavidin to sterically block the protein surface for inactivation. The caged protein was transduced into cells and reactivated by light-induced degradation of the conjugates. A cytotoxic protein, saporin, was caged and photoactivated both in vitro and in living cells with this method. This method achieved control of the cytotoxic activity in an off-on manner, introducing cell death selectively at the designed location using light. This simple and versatile photoactivation method is a promising tool for studying spatio-temporal cellular events that are related to intracellular proteins of interest.

Abstract 3291: Anti-HER2 scFv antibody-directed lentiviral delivery of myrosinase for eradicating HER2+ cancer cells after dosing prodrug sinigrin

Abstract Chemotherapy is widely used for the treatment of cancers, but it lacks selectivity and therefore results in collateral damage to healthy cells and drug resistance of cancer cells. Gene-directed enzyme prodrug therapy (GDEPT) has been adopted to mitigate these issues. In this study, we demonstrated the specific targeting of myrosinase-sinigrin paired GDEPT to treat HER2+ (human epidermal growth factor 2) cancer cells. Sinigrin excessively present in cruciferous vegetables can be catalyzed by myrosinase to produce cytotoxic ally isothiocyanate (AITC), which has shown anticancer effectiveness. The myrosinase gene was packed in biotinylated lentiviral vectors produced by co-transfection of HEK293T cells. The single chain variable fragment (scFv) gene sequence of anti-HER2 antibody and core streptavidin (coreSA) gene sequence were cloned in pET-30a(+) plasmid, which was then transformed in Lemo21 (DE3) competent cells for protein expression. The anti-HER2-coreSA chimeric protein was purified using immobilized metal affinity chromatography and characterized by SDS-PAGE and Western blot. The purified chimeric protein was tagged on biotinylated lentivirus via streptavidin-biotin binding. Our results showed that myrosinase-encoding lentivirus tagged with anti-HER2 selectively delivered myrosinase to HER2+ cancer cells. When treated with prodrug sinigrin, the targeted cancer cells underwent apoptosis and ensuing cell death. In summary, the targeted delivery of myrosinase to HER2+ cancer cells using lentivirus functionalized with anti-HER2 ScFv antibody can eradicate malignant cells when treated with enzymatically catalyzed prodrug sinigrin. Citation Format: Ammar Ahmad Tarar, Ching-An Peng. Anti-HER2 scFv antibody-directed lentiviral delivery of myrosinase for eradicating HER2+ cancer cells after dosing prodrug sinigrin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3291.

Unraveling Molecular Dynamics with High-Speed Video-Rate Atomic Force Microscopy

Abstract:This article describes the application of the fastest commercially available video-rate atomic force microscope (AFM) for studying the molecular dynamics of biological samples with a temporal resolution reaching 20 ms per frame. High-speed AFM was used for monitoring the kinetics of the temperature-induced ripple phase transition in supported lipid bilayers, DNA origami streptavidin-biotin binding, thermodynamic rehybridization of DNA, collagen type I fibrillogenesis, and the mobile annexin V trimer dynamics in 2D protein crystals. With no further need for sample processing, the applied method offers a breakthrough in molecular and sample dynamics measurements by enabling the real-time visualization of molecular processes and structural transitions at near-native conditions.

Rapid Capturing and Chemiluminescent Sensing of Programmed Death Ligand-1 Expressing Extracellular Vesicles

Cancer specific extracellular vesicles (EVs) are of significant clinical relevance, for instance programmed death ligand-1 (PD-L1) expressing EVs (PD-L1@EVs) have been shown to be ideal biomarker for non-invasive diagnosis of cancer and can predate the response of cancer patients to anti-PD-1/PD-L-1 immunotherapy. The development of sensitive and straightforward methods for detecting PD-L1@EVs can be a vital tool for non-invasive diagnosis of cancer. Most of the contemporary methods for EVs detection have limitations such as involvement of long and EV’s loss prone isolation methods prior to detection or they have employed expensive antibodies and instruments to accomplish detection. Therefore, we designed an ultracentrifugation-free and antibody-free sensing assay for PD-L1@EV by integrating Titanium oxide (TiO) coated magnetic beads (FeO@TiO) rapid capturing of EVs from undiluted serum with aptamers specificity and chemiluminescence (CL) sensitivity. To accomplish this we used FeO@TiO beads to rapidly capture EVs from the undiluted patient serum and added biotin labelled PD-L1 aptamer to specifically recognize PD-L1@EVs. Later, added streptavidin-modified Alkaline phosphates (ALP) taking advantage of biotin-streptavidin strong binding. Addition of CDP-star, a chemiluminescent substrate of ALP, initiates the chemiluminiscense that was recorded using spectrophotometer. The sensing assay showed high sensitivity with limit of detection (LOD) as low as EVs/mL and a wider linear correlation of CL intensity (a.u.) with the concentration of PD-L1@EVs from to EVs/mL. To examine the clinical utility of sensing assay we used undiluted serum samples from lung cancer patients and healthy individuals and successfully discern between healthy individuals and lung cancer patients. We are optimistic that the sensing assay can ameliorate our ability to be able to diagnose lung cancer non-invasively and can be helpful to predate the patient’s response to anti-PD-1/PD-L1 immunotherapy.

Synthesis of Spherical Nanoparticle Hybrids via Aerosol Thiol-Ene Photopolymerization and Their Bioconjugation.

Hybrid nanomaterials possess the properties of both organic and inorganic components and find applications in various fields of research and technology. In this study, aerosol photopolymerization is used in combination with thiol-ene chemistry to produce silver poly(thio-ether) hybrid nanospheres. In aerosol photopolymerization, a spray solution of monomers is atomized, forming a droplet aerosol, which then polymerizes, producing spherical polymer nanoparticles. To produce silver poly(thio-ether) hybrids, silver nanoparticles were introduced to the spray solution. Diverse methods of stabilization were used to produce stable dispersions of silver nanoparticles to prevent their agglomeration before the photopolymerization process. Successfully stabilized silver nanoparticle dispersion in the spray solution subsequently formed nanocomposites with non-agglomerated silver nanoparticles inside the polymer matrix. Nanocomposite particles were analyzed via scanning and transmission electron microscopy to study the degree of agglomeration of silver nanoparticles and their location inside the polymer spheres. The nanoparticle hybrids were then introduced onto various biofunctionalization reactions. A two-step bioconjugation process was developed involving the hybrid nanoparticles: (1) conjugation of (biotin)-maleimide to thiol-groups on the polymer network of the hybrids, and (2) biotin-streptavidin binding. The biofunctionalization with gold-nanoparticle-conjugates was carried out to confirm the reactivity of -SH groups on each conjugation step. Fluorescence-labeled biomolecules were conjugated to the spherical nanoparticle hybrids (applying the two-step bioconjugation process) verified by Fluorescence Spectroscopy and Fluorescence Microscopy. The presented research offers an effective method of synthesis of smart systems that can further be used in biosensors and various other biomedical applications.

Photo-Degradable Protein-Polymer Hybrid Shells for Caging Living Cells.

There is growing demand for the precise remote control of cellular functions in various fields. Herein, a method for caging mammalian cells by coating with photodegradable protein-polymer hybrid shells to photo-control their functions without genetic engineering is reported. A layer-by-layer assembly of photocleavable synthetic materials through biotin-streptavidin (SA) binding was employed for cell coating. The cell surfaces were first biotinylated with photocleavable biotinylated poly(ethylene glycol)(PEG)-lipid and then coated by repeatedly layering SA and micelles of the PEG-lipid and photocleavable biotinylated four-arm PEG. The cell extension and adhesion were suppressed with the shells and then triggered with the degradation of the shells by light exposure. Macrophage phagocytosis was also stopped by caging with the shells and restarted by light-guided uncaging. This study provides the first proof of principle that cellular functions can be remotely controlled by steric hinderance of cell surfaces with photodegradable materials.

An Aptamer-Array-Based Sample-to-Answer Biosensor for Ochratoxin A Detection via Fluorescence Resonance Energy Transfer

Food toxins are a hidden threat that can cause cancer and tremendously impact human health. Therefore, the detection of food toxins in a timely manner with high sensitivity is of paramount importance for public health and food safety. However, the current detection methods are relatively time-consuming and not practical for field tests. In the present work, we developed a novel aptamer-chip-based sample-to-answer biosensor (ACSB) for ochratoxin A (OTA) detection via fluorescence resonance energy transfer (FRET). In this system, a cyanine 3 (Cy3)-labeled OTA-specific biotinylated aptamer was immobilized on an epoxy-coated chip via streptavidin-biotin binding. A complementary DNA strand to OTA aptamer at the 3′-end was labeled with a black hole quencher 2 (BHQ2) to quench Cy3 fluorescence when in proximity. In the presence of OTA, the Cy3-labeled OTA aptamer bound specifically to OTA and led to the physical separation of Cy3 and BHQ2, which resulted in an increase of fluorescence signal. The limit of detection (LOD) of this ACSB for OTA was 0.005 ng/mL with a linearity range of 0.01–10 ng/mL. The cross-reactivity of ACSB against other mycotoxins, ochratoxin B (OTB), aflatoxin B1 (AFB1), zearalenone (ZEA), or deoxynilvalenol (DON), was less than 0.01%. In addition, this system could accurately detect OTA in rice samples spiked with OTA, and the mean recovery rate of the spiked-in OTA reached 91%, with a coefficient of variation (CV) of 8.57–9.89%. Collectively, the ACSB may represent a rapid, accurate, and easy-to-use platform for OTA detection with high sensitivity and specificity.

Heparin–Avastin Complexes Show Enhanced VEGF Binding and Inhibition of VEGF-Mediated Cell Migration

Bevacizumab (known by the tradename Avastin) is an antibody that binds VEGF and blocks its binding to VEGF receptors on endothelial cells, and is used to treat cancers and other diseases associated with excessive vascular growth. Our previous findings showed enhanced VEGF binding to Avastin in the presence of heparin, indicating that colocalizing heparin with Avastin could enhance VEGF inhibitory activity. Thus, the aim of the present study was to determine if conjugating Avastin and heparin to one another would lead to enhanced anti-VEGF activity. Avastin was conjugated to either biotin or streptavidin, and biotin–heparin was used to bring the two molecules into close proximity via biotin–streptavidin binding. Radioligand binding assays with 125 I-VEGF and cell migration assays using human umbilical vein endothelial cells were used to evaluate the impact of heparin on Avastin binding and activity. We found that bringing Avastin and heparin together, either on a surface or through streptavidin conjugation of Avastin, led to increased VEGF binding compared to that with each molecule alone. The heparin-mediated increase in VEGF binding was also noted at acidic pH where Avastin showed decreased VEGF binding. Conditions where Avastin and heparin showed enhanced VEGF binding also showed reduced VEGF-induced migration of human umbilical vein endothelial cells. These findings suggest design principles for a modified Avastin-based inhibitor of angiogenesis.