Biotin-functionalized nanoparticles: an overview of recent trends in cancer detection.

Electrochemical bio-sensing is a potent and efficient method for converting various biological recognition events into voltage, current, and impedance electrical signals. Biochemical sensors are now a common part of medical applications, such as detecting blood glucose levels, detecting food pathogens, and detecting specific cancers. As an exciting feature, bio-affinity couples, such as proteins with aptamers, ligands, paired nucleotides, and antibodies with antigens, are commonly used as bio-sensitive elements in electrochemical biosensors. Biotin-avidin interactions have been utilized for various purposes in recent years, such as targeting drugs, diagnosing clinically, labeling immunologically, biotechnology, biomedical engineering, and separating or purifying biomolecular compounds. The interaction between biotin and avidin is widely regarded as one of the most robust and reliable noncovalent interactions due to its high bi-affinity and ability to remain selective and accurate under various reaction conditions and bio-molecular attachments. More recently, there have been numerous attempts to develop electrochemical sensors to sense circulating cancer cells and the measurement of intracellular levels of protein thiols, formaldehyde, vitamin-targeted polymers, huwentoxin-I, anti-human antibodies, and a variety of tumor markers (including alpha-fetoprotein, epidermal growth factor receptor, prostate-specific Ag, carcinoembryonic Ag, cancer antigen 125, cancer antigen 15-3, etc.). Still, the non-specific binding of biotin to endogenous biotin-binding proteins present in biological samples can result in false-positive signals and hinder the accurate detection of cancer biomarkers. This review summarizes various categories of biotin-functional nanoparticles designed to detect such biomarkers and highlights some challenges in using them as diagnostic tools.

Acoustic detection of a mutation-specific Ligase Chain Reaction based on liposome amplification.

Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based amplification and detection techniques, such as Rolling Circle Amplification and Ligase Detection Reaction, suffer from low efficiency and difficulties in product detection. To address these limitations, we propose a novel approach that combines Ligase Chain Reaction (LCR) with acoustic detection using highly dissipative liposomes. In our study, we are using LCR combined with biotin- and cholesterol-tagged primers to produce amplicons also modified at each end with a biotin and cholesterol molecule. We then apply the LCR mix without any purification directly on a neutravidin modified QCM device Au-surface, where the produced amplicons can bind specifically through the biotin end. To improve sensitivity, we finally introduce liposomes as signal enhancers. For demonstration, we used the detection of the BRAF V600E point mutation versus the wild-type allele, achieving an impressive detection limit of 220 aM of the mutant target in the presence of the same amount of the wild type. Finally, we combined the assay with a microfluidic fluidized bed DNA extraction technology, offering the potential for semi-automated detection of SNVs in patients' crude samples. Overall, our LCR/acoustic method outperforms other LCR-based approaches and surface ligation biosensing techniques in terms of detection efficiency and time. It effectively overcomes challenges related to DNA detection, making it applicable in diverse fields, including genetic disease and pathogen detection.

All-stage targeted red blood cell membrane-coated docetaxel nanocrystals for glioma treatment.

Challenges for glioma treatment with nanomedicines include physio-anatomical barriers (the blood-brain barrier and blood-brain tumor barrier), low drug loading capacity, and limited circulation time. Here, a red blood cell membrane-coated docetaxel drug nanocrystal (pV-RBCm-NC(DTX)), modified with pHA-VAP (pV) for all-stage targeting of glioma, was designed. The NC(DTX) core exhibited a high drug loading capacity but low in vivo stability, and the RBCm coating significantly enhanced the stability and prolonged in vivo circulation. Moreover, the Y-shaped targeting ligand pV was modified by a mild avidin-biotin interaction, which endowed RBCm-NC(DTX) with superior barrier-crossing ability and therapeutic efficacy. The integration of nanocrystal technology, cell membrane coating, and the avidin-biotin insertion method into this active targeting biomimetic formulation represents a promising drug delivery strategy for glioma.

Live-cell imaging of human apurinic/apyrimidinic endonuclease 1 in the nucleus and nucleolus using a chaperone@DNA probe.

Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear APE1 in live cells remains challenging. Here, we report a chaperone@DNA probe for live-cell imaging of APE1 in the nucleus and nucleolus in real time. The probe is based on an assembly of phenylboronic acid modified avidin and biotin-labeled DNA containing an abasic site (named PB-ACP), which cleverly protects DNA from being nonspecifically destroyed while enabling targeted delivery of the probe to the nucleus. The PB-ACP construct specifically detects APE1 due to the high binding affinity of APE1 for both avidin and the abasic site in DNA. It is easy to prepare, biocompatible and allowing for long-term observation of APE1 activity. This molecular tool offers a powerful means to investigate the behavior of APE1 in the nuclei of various types of live cells, particularly for the development of improved cancer therapies targeting this protein.

Macrophage membrane (MMs) camouflaged near-infrared (NIR) responsive bone defect area targeting nanocarrier delivery system (BTNDS) for rapid repair: promoting osteogenesis via phototherapy and modulating immunity.

Bone defects remain a significant challenge in clinical orthopedics, but no targeted medication can solve these problems. Inspired by inflammatory targeting properties of macrophages, inflammatory microenvironment of bone defects was exploited to develop a multifunctional nanocarrier capable of targeting bone defects and promoting bone regeneration. The avidin-modified black phosphorus nanosheets (BP-Avidin, BPAvi) were combined with biotin-modified Icaritin (ICT-Biotin, ICTBio) to synthesize Icaritin (ICT)-loaded black phosphorus nanosheets (BPICT). BPICT was then coated with macrophage membranes (MMs) to obtain MMs-camouflaged BPICT (M@BPICT). Herein, MMs allowed BPICT to target bone defects area, and BPICT accelerated the release of phosphate ions (PO43-) and ICT when exposed to NIR irradiation. PO43- recruited calcium ions (Ca2+) from the microenvironment to produce Ca3(PO4)2, and ICT increased the expression of osteogenesis-related proteins. Additionally, M@BPICT can decrease M1 polarization of macrophage and expression of pro-inflammatory factors to promote osteogenesis. According to the results, M@BPICT provided bone growth factor and bone repair material, modulated inflammatory microenvironment, and activated osteogenesis-related signaling pathways to promote bone regeneration. PTT could significantly enhance these effects. This strategy not only offers a solution to the challenging problem of drug-targeted delivery in bone defects but also expands the biomedical applications of MMs-camouflaged nanocarriers.

Combining avidin with CD63 improves basophil activation test accuracy in classifying peanut allergy.

BACKGROUND: Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore-labeled avidin, a positively-charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. METHODS: Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01-10,000 ng/mL) and four control conditions (no stimulant, anti-IgE, fMLP (N-formylmethionine-leucyl-phenylalanine), and anti-FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. RESULTS: Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+ /CD63- cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. CONCLUSIONS: Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy.

Inhibiting Multidrug Resistance with Transferrin-Targeted Polymersomes through Optimization of Ligand Density.

Transferrin-conjugated polymersomes, transferrin-biotin/avidin/biotin-Pluronic F127-poly(lactic acid) (Tf-F127-PLA), were successfully prepared through a biotin-avidin bridging technique to study their ability to inhibit multidrug resistance of cancer cells. Hydrophilic doxorubicin (DOX) was selected as the model drug to be loaded into Tf-F127-PLA polymersomes. DOX loaded in Tf-F127-PLA polymersomes was released fast initially, followed by a slow release. The effect of the transferrin ligand density of Tf-F127-PLA/DOX polymersomes on their targeting properties was studied by both cytotoxicity and cellular uptake assays against A549 lung cancer cells. It was shown that Tf-F127-PLA/DOX polymersomes had better targeting ability than nontargeted drug-loaded polymersomes. Furthermore, Tf-F127-PLA/DOX polymersomes with 2% Tf molar content have more effective antitumor activity and a higher cellular uptake than those with 4 and 5% Tf molar content. 2% Tf-F127-PLA/DOX polymersomes also exhibited better anticancer ability in multidrug resistant cancer cells A549/ADR than nontargeted PLA-F127-PLA/DOX polymersomes. It was further proved that the endocytosis of polymersomes by A549/ADR cells was an energy-dependent endocytosis process, which was related to clathrin, macrocytosis, and caveolin. Also, the endocytosis of Tf-F127-PLA/DOX polymersomes was proven to be mediated by the transferrin receptor.

PD-L1 ImmunoPET on the basis of Avidin/Biotin pre-targeted cancer imaging.

This study aimed to establish the radio-immune imaging protocol on the basis of Avidin/Biotin system. The programmed death-ligand 1 (PD-L1) antibody (Atezolizumab) was employed as the primary molecule in targeting PD-L1, and the two-step strategy, consisting of the first injection of Avidin-conjugated PD-L1 monoclonal antibody (Atezolizumab) and the second injection of 7.4 MBq 68Ga-Biotin with a 60 h interval, was then verified on the colon cancer-bearing mice. PET imaging was performed at 30, 90, 180 min to measure the standard uptake value and tumor to liver ratios. Cellular binding experiments and in vivo distribution showed that the conjugation of Avidin did not affect the affinity of Atezolizumab to PD-L1 antigen. Biotin was radio-labeled with 68Ga with radiolabeling efficiency of 70.5 ± 3.5% and purification was needed to increase the radiochemical purity. For PD-L1-positive tumors, SUVmax was 0.38 ± 0.06 in the Avidin-Atezolizumab pre-treated mice at 90 min; the tumor/liver ratios of pre-targeting group were 1.06 ± 0.19 and 0.97 ± 0.16 at 30 and 90 min, while the absence of pre-treatment of Avidin was of the lower ratios as 0.88 ± 0.01 and 0.54 ± 0.11 when 68Ga-Biotin served as the radiopharmaceutical as well. In conclusion, pre-targeting immunoPET strategy can elevate the target-to-nontarget ratio, decrease the blood background and shorten the interval between injection of radiopharmaceuticals and PET scan, providing a highly PD-L1-specific and sensitive imaging method for the detection of tumorous immune micro-environment.

Biotin receptor-mediated intracellular delivery of synthetic polypeptide-protein complexes.

Pulmonary delivery offers a non-invasive route for the administration of biotherapeutics. In this context, understanding and control of a transport into, and across cellular barriers is central to the design of delivery systems. Here, we report our study on receptor mediated delivery of protein cargo by a formulation comprising sub-300 nm sized non-covalent protein complexes with biotin-conjugated PEG-poly(glutamic acid) (biotin-PEG2k-b-GA10) and PEG2k-b-GA30 copolymers blend as targeting and complexing functionalities. Designed complexes achieve intracellular delivery of the cargo in lung derived A549 epithelial cells in vitro via sodium-dependent multivitamin transporter (biotin receptor). We further show that biotin receptor driven endocytosis preferentially involves dynamin- and caveolae-dependent vesicular internalization, switching the transport pathway away from predominantly clathrin-dependent entry of free protein. Significantly for a protective intracellular delivery of biotherapeutics based on non-covalent complexation with polymeric excipients, the study provides evidence of intracellular presence of the complexing copolymer; demonstrated exploiting biotin in biotin-PEG2k-b-GA10 copolymer as a tag for binding with fluorescently labelled avidin. Moreover, analysis of intracellular localization of constitutive species shortly following cellular internalization suggests a co-localization of biotin-PEG2k-b-GA10 copolymer and protein constitutive species. The study demonstrates intracellular delivery of biotin targeted non-covalent complexes with a protein cargo, the result with important implications in a design of enabling technology platforms for protective, receptor mediated intracellular delivery of biotherapeutics.

Sustained intra-cartilage delivery of interleukin-1 receptor antagonist using cationic peptide and protein-based carriers.

OBJECTIVE: Blocking the interleukin-1 (IL-1) catabolic cascade following joint trauma can be achieved using its receptor antagonist, IL-1Ra. However, its clinical translation for osteoarthritis therapy has been unsuccessful due to its rapid joint clearance and lack of targeting and penetration into deep cartilage layers at therapeutic concentrations. Here, we target the high negative charge of cartilage aggrecan-glycosaminoglycans (GAGs) by attaching cationic carriers to IL-1Ra. IL-1Ra was conjugated to the cartilage targeting glycoprotein, Avidin, and a short length optimally charged cationic peptide carrier (CPC+14). It is hypothesized that electro-diffusive transport and binding properties of IL-1Ra-Avidin and IL-1Ra-CPC+14 will create intra-cartilage depots of IL-1Ra, resulting in long-term suppression of IL-1 catabolism with only a single administration. DESIGN: IL-1Ra was conjugated to Avidin or CPC+14 using site specific maleimide linkers, and confirmed using gel electrophoresis, high-performance liquid chromatography (HPLC), and mass spectrometry. Intra-cartilage transport and retention of conjugates was compared with native IL-1Ra. Attenuation of IL-1 catabolic signaling with one-time dose of IL-1Ra-CPC+14 and IL-1Ra-Avidin was assessed over 16 days using IL-1α challenged bovine cartilage and compared with unmodified IL-1Ra. RESULTS: Positively charged IL-1Ra penetrated through the full-thickness of cartilage, creating a drug depot. A single dose of unmodified IL-1Ra was not sufficient to attenuate IL-1-induced cartilage deterioration over 16 days. However, when delivered using Avidin, and to a greater extent CPC+14, IL-1Ra significantly suppressed cytokine induced GAG loss and nitrite release while improving cell metabolism and viability. CONCLUSION: Charge-based cartilage targeting drug delivery systems hold promise as they can enable long-term therapeutic benefit with only a single dose.

Preliminary efficacy of [90Y]DOTA-biotin-avidin radiotherapy against non-muscle invasive bladder cancer.

PURPOSE: Bladder cancer represents 3% of all new cancer diagnoses per year. We propose intravesical radionuclide therapy using the ß-emitter 90Y linked to DOTA-biotin-avidin ([90Y]DBA) to deliver short-range radiation against non-muscle invasive bladder cancer (NMIBC). MATERIAL AND METHODS: Image-guided biodistribution of intravesical DBA was investigated in an animal model by radiolabeling DBA with the 68Ga and dynamic microPET imaging following intravesical infusion of [68Ga]DBA for up to 4 h and post-necropsy γ-counting of organs. The antitumor activity of [90Y]DBA was investigated using an orthotopic MB49 murine bladder cancer model. Mice were injected with luciferase-expressing MB49 cells and treated via intravesical administration with 9.2 MBq of [90Y]DBA or unlabeled DBA 3 days after the tumor implantation. Bioluminescence imaging was conducted after tumor implantation to monitor the bladder tumor growth. In addition, we investigated the effects of [90Y]DBA radiation on urothelial histology with immunohistochemistry analysis of bladder morphology. RESULTS: Our results demonstrated that DBA is contained in the bladder for up to 4 h after intravesical infusion. A single dose of [90Y]DBA radiation treatment significantly reduced growth of MB49 bladder carcinoma. Attaching 90Y-DOTA-biotin to avidin prevents its re-absorption into the blood and distribution throughout the rest of the body. Furthermore, immunohistochemistry demonstrated that [90Y]DBA radiation treatment did not cause short-term damage to urothelium at day 10, which appeared similar to the normal urothelium of healthy mice. CONCLUSION: Our data demonstrates the potential of intravesical [90Y]DBA as a treatment for non-muscle invasive bladder cancer.

Early Assessment of Atherosclerotic Lesions and Vulnerable Plaques in vivo by Targeting Apoptotic Macrophages with AV Nanobubbles.

Background: The early detection of atherosclerotic lesions is particularly important for risk prediction of acute cardiovascular events. Macrophages apoptosis was significantly associated with the degree of AS lesions and especially contributed to plaque vulnerability. In this research, we mainly sought to explore the feasibility of a home-made AV-nanobubbles (NBAV) for visualization of apoptotic macrophages and assessment of atherosclerosis (AS) lesions by contrast-enhanced ultrasound (CEUS) imaging. Methods: NBAV were prepared by "Optimized Thin-Film Hydration" and "Biotin-Avidin-Biotin" methods. Then, the characterization and echogenicity of NBAV were measured and analyzed in vitro. The targeting ability of NBAV to ox-LDL-induced apoptotic macrophages was observed by laser scanning confocal microscope. The ApoE-/- mice mode fed with high fat diet were observed by high-frequency ultrasound, microanatomy and oil red O staining. CEUS imaging in vivo was performed on AS plaques with NBAV and NBCtrl injection through the tail vein in turn in ApoE-/- mice. After CEUS imaging, the plaques were confirmed and analyzed by histopathological and immunological assessment. Results: The prepared NBAV had a nano-scale size distribution with a low PDI and a negative zeta potential. Moreover, NBAV showed an excellent stability and exhibited a significantly echogenic signal than saline in vitro. In addition, we found that NBAV could target apoptotic macrophages induced by ox-LDL. Compared with NBCtrl, CEUS imaging of NBAV showed strong and sustained echo enhancement in plaque area of aortic arch in vivo. Further research showed that NBAV sensitive plaques presented more significant pathological changes with several vulnerable plaque features and abundant TUNEL-positive area. Conclusion: NBAV displayed a sensitive indicator to evaluate apoptotic macrophages, indicating a promising CEUS molecular probe for AS lesions and vulnerable plaques identification.

A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions.

Receptor-ligand binding has been analyzed at the protein level using isothermal titration calorimetry and surface plasmon resonance and at the cellular level using interaction-associated downstream gene induction/suppression. However, no currently available technique can characterize this interaction directly through visualization. In addition, all available assays require a large pool of cells; no assay capable of analyzing receptor-ligand interactions at the single-cell level is publicly available. Here, we describe a new microfluidic chip-based technique for analyzing and visualizing these interactions at the single-cell level. First, a protein is immobilized on a glass slide and a low-flow-rate pump is used to isolate cells that express receptors that bind to the immobilized ligand. Specifically, we demonstrate the efficacy of this technique by immobilizing biotin-conjugated FGL2 on an avidin-coated slide chip and passing a mixture of GFP-labeled wild-type T cells and RFP-labeled FcγRIIB-knockout T cells through the chip. Using automated scanning and counting, we found a large number of GFP+ T cells with binding activity but significantly fewer RFP+ FcγRIIB-knockout T cells. We further isolated T cells expressing a membrane-anchored, tumor-targeted IL-12 based on the receptor's affinity to vimentin to confirm the versatility of our technique. This protocol allows researchers to isolate receptor-expressing cells in about 4 hours for further downstream processing.

Ion-selective electrode-based potentiometric immunoassays for the quantitative monitoring of alpha-fetoprotein by coupling rolling cycle amplification with silver nanoclusters.

Potentiometric immunoassays have been utilized for the quantitative detection of alpha-fetoprotein (AFP) in hepatocellular carcinoma, but most of them involve low sensitivity and enzyme labels, and thus are unfavorable for routine use. In this work, we report the proof-of-concept of a sensitive and powerful ion-selective potentiometric sensing method for AFP detection with an in situ amplified signal readout. This potentiometric immunoassay mainly contains a silver nanocluster-functionalized single-stranded DNA (AgNC-DNA), a short DNA primer and two antibodies. A sandwich-type immunoreaction is employed for AFP determination on an anti-AFP capture antibody-coated microplate using a biotinylated human AFP secondary antibody. Coupling with a typical biotin-avidin system, the biotinylated DNA initiator strands are conjugated on the microplate in the presence of AFP to induce the rolling cycle amplification (RCA) reaction, followed by AgNC-DNA hybridization. Upon addition of HNO3, the hybridized AgNCs are dissolved into numerous Ag(I) ions, which can be readily determined on a portable handheld silver-ion selective electrode (Ag-ISE). Under optimal conditions, the electrode potential increases with an increase in AFP concentration and exhibits a good linear range of 0.01-100 ng mL-1 at a detection limit of 7.9 pg mL-1. Moreover, the Ag-ISE-based potentiometric immune assay also shows good reproducibility, high specificity and long-term storage stability. Importantly, 18 human serum specimens containing the AFP analyte are screened using the potentiometric immunoassay, giving well-matched experimental results relative to the referenced enzyme-linked immunosorbent assay (ELISA) method.

pH-Sensitive Twin Liposomes Containing Quercetin and Laccase for Tumor Therapy.

In this study, functional twin liposomes (TLs) were designed by linking avidin-anchored single liposomes and biotin-anchored single liposomes via avidin-biotin interactions. Here, we first punched a hole on the liposome surface using the liposome magnetoporation method to prepare functional single liposomes, which were used for safely encapsulating quercetin (QER, as a model prodrug) or laccase (LAC, as a bioactive enzyme) inside the liposomes without the use of organic solvents; the pores were then plugged by pH-sensitive glycol chitosan grafted with 3-diethylaminopropylamine (GDEAP) and avidin (or biotin). As a result, single liposomes with QER and biotin-GDEAP were efficiently coupled with other liposomes with LAC and avidin-GDEAP. We demonstrated that the TLs could accelerate QER and LAC release at acidic pH (6.8), improving the LAC-mediated oxidization of QER and significantly elevating tumor cell death, suggesting that this strategy can be used as an efficient method for the programmed action of prodrugs.

Antigen-Down PEC Immunosensor for CYFRA21-1 Detection Based on Photocurrent Polarity Switching Strategy.

In this work, an antigen-down photoelectrochemical (PEC) immunosensor based on a signal polarity switching strategy for the detection of cytokeratin 19 fragment 21-1 (CYFRA21-1) was proposed. 3,4,9,10-Perylene tetracarboxylic acid (PTCA) is a conjugated organic dye containing five benzene nuclei, which has excellent film-forming and optical properties. PTCA sensitized by SnS2 can further improve the basal signal and the stability of the PEC immunosensor. Moreover, avidin-functionalized CuInS2 as a signal probe can convert the basal anodic photocurrent to a cathodic photocurrent. Therefore, the PEC sensor realized the photocurrent polarity conversion before and after labeling. With avidin-functionalized CuInS2, the polarity of the photocurrent was changed once CYFRA21-1 was detected. Therefore, the PEC immunosensor owns high sensitivity. The linear range of the immunosensor for the detection of CYFRA21-1 is 0.00001-500 ng·mL-1, and the detection limit is 3.5 fg·mL-1. The PEC immunosensor has good stability, high selectivity, and good repeatability. This work may provide a new way for the detection of CYFRA21-1 and other proteins.

Probing the leak pathway: Live-cell imaging of macromolecule passage through epithelia.

Epithelia compartmentalize multicellular organisms and provide interfacing between the inside and outside. Apart from regulating the exchange of solutes, uptake of nutrients, and excretion of waste products, their major function is to prevent uncontrolled access of foreign material to immune-competent compartments. Progress in understanding this barrier function toward larger solutes and its possible defects, as can be seen in a variety of diseases, is largely hampered by a lack of methods to spatiotemporally resolve transepithelial passage of macromolecules. Using different cell culture epithelia, we applied biotinylated dextran tracers carrying an acceptor fluorophore. These bind to cell-adherent avidin carrying donor fluorophore at the basolateral membranes of single-layered epithelial sheets. Confocal fluorescence microscopy was applied to living epithelia in order to image apical-to-basolateral tracer passage as a Förster resonance energy transfer signal of the fluorescent dextran-avidin pair over time. Stimulated macromolecule passage using barrier-perturbing agents proved its effectiveness for the leak imaging method presented herein. Over hours of imaging, spontaneous leaks were rare, occurring transiently on the scale of minutes and for the most part associated with rearranging cell junctions. The discussed approach to leak imaging is expected to promote the understanding of epithelial barriers, particularly, the nature and dynamics of the epithelial cell leak pathway.

The Effect of Avidin on Viability and Proliferation of Colorectal Cancer Cells HT-29.

OBJECTIVE: The aim of this study was to analyze the effect of avidin treatment on cell viability, proliferation and cyclin D1 expression in colorectal cancer cells HT-29. METHODS: Colorectal cancer cell line HT-29 incubated with 50, 100, 150, and 200 µg/mL of avidin concentration during 24, 48, and 72 hours, then the cell viability and proliferation   were analyzed. Each avidin concentration was conducted together with HT-29 cell line without avidin treatment as a control group. The cell viability was measured by MTS assay and the proliferation was measured by BrdU (5-bromo-2'-deoxyuridine) cell proliferation assay. According to cell viability and proliferation result, we determined the 100 µg/mL avidin concentration for analyzing mRNA and protein of cyclin D1. RESULTS: We demonstrated that the viability and proliferation of HT-29 cells were significantly decreased in all concentration of avidin treatment compared to control.   The cell proliferation showed larger reduction in avidin treatment rather than cell viability. This proves avidin could inhibit proliferation of colorectal cancer cell HT-29 quite well. The expression of cyclin D1, both mRNA and protein, was also significantly decreased after the avidin treatment group compared to control group, it supports the suppression of proliferation result. CONCLUSION: We concluded that avidin treatment could decrease cell viability and proliferation, accompanied by suppression of cyclin D1 expression in colorectal cells HT-29.

Detection of Lung Cancer Cells in Solutions Using a Terahertz Chemical Microscope.

Cancer genome analysis has recently attracted attention for personalized cancer treatment. In this treatment, evaluation of the ratio of cancer cells in a specimen tissue is essential for the precise analysis of the genome. Conventionally, the evaluation takes at least two days and depends on the skill of the pathologist. In our group, a terahertz chemical microscope (TCM) was developed to easily and quickly measure the number of cancer cells in a solution. In this study, an antibody was immobilized on a sensing plate using an avidin-biotin reaction to immobilize it for high density and to improve antibody alignment. In addition, as the detected terahertz signals vary depending on the sensitivity of the sensing plate, the sensitivity was evaluated using pH measurement. The result of the cancer cell detection was corrected using the result of pH measurement. These results indicate that a TCM is expected to be an excellent candidate for liquid biopsies in cancer diagnosis.

Surface Plasmon Resonance for Protease Detection by Integration of Homogeneous Reaction.

The heterogeneous assays of proteases usually require the immobilization of peptide substrates on the solid surface for enzymatic hydrolysis reactions. However, immobilization of peptides on the solid surface may cause a steric hindrance to prevent the interaction between the substrate and the active center of protease, thus limiting the enzymatic cleavage of the peptide. In this work, we reported a heterogeneous surface plasmon resonance (SPR) method for protease detection by integration of homogeneous reaction. The sensitivity was enhanced by the signal amplification of streptavidin (SA)-conjugated immunoglobulin G (SA-IgG). Caspase-3 (Cas-3) was determined as the model. A peptide labeled with two biotin tags at the N- and C-terminals (bio-GDEVDGK-bio) was used as the substrate. In the absence of Cas-3, the substrate peptide was captured by neutravidin (NA)-covered SPR chip to facilitate the attachment of SA-IgG by the avidin-biotin interaction. However, once the peptide substrate was digested by Cas-3 in the aqueous phase, the products of bio-GDEVD and GK-bio would compete with the substrate to bond NA on the chip surface, thus limiting the attachment of SA-IgG. The method integrated the advantages of both heterogeneous and homogeneous assays and has been used to determine Cas-3 inhibitor and evaluate cell apoptosis with satisfactory results.