Touchpad-based immunochromatographic strip for detecting the skin surface proteins.

This study demonstrates, for the first time, the proof-of-concept of a novel immunosensor, a touchpad-based immunochromatographic strip, that non-invasively extracts and detects skin surface proteins. The strip was composed of a nitrocellulose membrane at the center, where a spot of anti-human IgG capture antibody was physically adsorbed. The capture antibody spot was covered with a glass fiber membrane impregnated with phosphate-buffered saline (PBS) to extract skin surface proteins, avoiding direct contact of the human skin with the capture antibodies. Skin surface IgG was detected in two steps: (1) touching the capture antibody via a glass fiber membrane containing PBS, and (2) dipping the strip into the Au-nanoparticle-labeled secondary antibody to visualize the existence of the captured skin surface IgG on the strip. We qualitatively demonstrated that using a very small amount of PBS while maintaining contact with the skin, skin surface proteins can be concentrated and detected, even with a relatively low-sensitivity immunochromatographic chip. This sensor is expected to be a potential biosensor for the non-invasive diagnosis of the integrity of human skin.

Enhancing Single-Cell Western Blotting Sensitivity Using Diffusive Analyte Blotting and Antibody Conjugate Amplification.

While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein content often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity Western blots on single cells (scWesterns) are attractive because they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by the affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address the sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in the limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates, which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 1000 molecules, a 120-fold improvement. This enables us to detect 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies compared to 84.5% of cells using in-gel detection. These results suggest the compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents─not previously accessible for in-gel use─for further signal amplification and detection of low-abundance targets.

13 CO2 labelling as a tool for elucidating the mechanism of cuticle development: a case of Clusia rosea.

The plant cuticle is an important plant-atmosphere boundary, the synthesis and maintenance of which represents a significant metabolic cost. Only limited information regarding cuticle dynamics is available. We determined the composition and dynamics of Clusia rosea cuticular waxes and matrix using 13 CO2 labelling, compound-specific and bulk isotope ratio mass spectrometry. Collodion was used for wax collection; gas exchange techniques to test for any collodion effects on living leaves. Cutin matrix (MX) area density did not vary between young and mature leaves and between leaf sides. Only young leaves incorporated new carbon into their MX. Collodion-based sampling discriminated between epicuticular (EW) and intracuticular wax (IW) effectively. Epicuticular differed in composition from IW. The newly synthetised wax was deposited in IW first and later in EW. Both young and mature leaves synthetised IW and EW. The faster dynamics in young leaves were due to lower wax coverage, not a faster synthesis rate. Longer-chain alkanes were deposited preferentially on the abaxial, stomatous leaf side, producing differences between leaf sides in wax composition. We introduce a new, sensitive isotope labelling method and demonstrate that cuticular wax is renewed during leaf ontogeny of C. rosea. We discuss the ecophysiological significance of the new insights.

Heat-induced antigen retrieval utilizing modified Tris-EDTA buffer for reprobing of Western blots on nitrocellulose paper.

The development of heat-induced antigen retrieval technologies with Tris-EDTA buffer has dramatically improved immunostaining of specific antigens for routine immunohistochemical detection (Krenacs et al., 2010) [1]. However, little evidence exists on whether heat-Induced antigen retrieval utilizing Tris-EDTA buffer can strip western blot (WB) membranes and allow sequential reprobing. Here, we serendipitously discover that ∼95 °C Tris-EDTA buffer with 0.01% Tween 20 could repeatedly strip the Nitrocellulose membranes (NC). After electroblotting, NC blots were soaked into Tris-EDTA stripping buffer (∼95 °C, 10-25min) and we could perform at least five rounds (the following antibodies used: Vinculin, Atg7, Caspase-3, UBA5, JNK and ERK1/2) stripping in sequential chemiluminescent detections. The NC membranes also show clear western signals and background without losing transferred proteins during the reprobing process of WB. Hence, this study report additional new roles of the heat-Induced antigen retrieval Tris-EDTA buffer with 0.01% Tween 20. The method is simpler, more affordable and harmless for the nitrocellulose paper, which will be helpful for effective reprobing in western blotting applications.

Wet nitrocellulose membrane for the level 3 feature visualization of various latent fingerprints and gender determination.

A facile and high-resolution enhancement of latent fingerprints (LFPs) has been developed by using a wet nitrocellulose (NC) membrane as a matrix under natural light. A clear fingerprint pattern was presented on the membrane after a fingertip touch owing to the difference in light transmittance between the ridge residues and the wet-NC-membrane background. Compared with conventional methods, this protocol can provide a higher resolution fingerprint image to extract level 3 details accurately. It is also compatible with commonly used fingerprint visualization techniques (magnetic ferric oxide powder and AgNO3. The modified membrane could be more general to realize the high-resolution visualization of LFP transferred from various substrates, even independent of light projection. Due to the excellent feasibility and reproducibility of level 3 details extracted by the wet NC membrane, the frequency distribution of the distance between adjacent sweat pores (FDDasp) could be used to effectively distinguish the fragmentary fingerprints. Finally, the level 3 features of LFPs from females and males were conveniently extracted by the wet-NC-membrane method for gender identification. The statistical results indicated that females had a higher average sweat pore density (115/9 mm2) than males (84/9 mm2). Taken together, this approach provided a high-resolution, reproducible, and accurate imaging of LFPs, which shows great promise for forensic information analysis.

Impact of filter material and holding time on spore sampling efficiency in water.

Bacillus anthracis and other environmentally persistent pathogens pose a significant threat to human and environmental health. If contamination is spread over a wide area (e.g. resulting from a bioterrorism or biowarfare incident), readily deployable and scalable sample collection methods will be necessary for rapidly developing and implementing effective remediation strategies. A recent surge in environmental (eDNA) sampling technologies could prove useful for quantifying the extent and levels of contamination from biological agents in environmental and drinking water. In this study, three commonly used membrane filtration materials (cellulose acetate, cellulose nitrate, and nylon) were evaluated for spore filtration efficiency, yielding recoveries from 17%-68% to 25%-117% for high and low titer samples, respectively, where cellulose nitrate filters generated the highest recoveries. A holding time test revealed no statistically significant differences between spore recoveries when analyzed at the specified timepoints, suggesting that eDNA filter sampling techniques can yield and maintain a relatively high recovery of spores for an extended period of time between filtration and analysis without a detrimental impact on spore recoveries. The results shown here indicate that emerging eDNA technologies could be leveraged for sampling following a wide-area contamination incident and for other microbiological water sampling applications.

Acral collodion membrane associated with ichthyosis due to a heterozygous pathogenic variant of ELOVL4 gene.

A female twin presented at birth with a collodion membrane on the hands and feet. After the membrane resolved over the first months of life, she was initially diagnosed with acral self-healing collodion membrane. However, she subsequently developed brown well-defined geometric scales on the trunk and extremities, consistent with ichthyosis. Genetic testing showed a heterozygous pathogenic variant in ELOVL4, a gene associated with syndromic ichthyosis with developmental delay, seizures, and spasticity. Although acral collodion membrane is considered to be a benign variant of the more generalized collodion, usually described as "self-healing," it may be the initial presentation of more diffuse ichthyosis.

Graphene Oxide/Nitrocellulose Non-Covalent Hybrid as Solid Phase for Oligo-DNA Extraction from Complex Medium.

A nitrocellulose-graphene oxide hybrid that consists of a commercially nitrocellulose (NC) membrane non-covalently modified with graphene oxide (GO) microparticles was successfully prepared for oligonucleotide extraction. The modification of NC membrane was confirmed by Fourier Transform Infrared Spectroscopy (FTIR), which highlighted the principal absorption bands of both the NC membrane at 1641, 1276, and 835 cm-1 (NO2) and of GO in the range of 3450 cm-1 (CH2-OH). The SEM analysis underlined the well-dispersed and uniform coverage of NC membrane with GO, which displayed thin spider web morphology. The wettability assay indicated that the NC-GO hybrid membrane exhibited slightly lower hydrophilic behavior, with a water contact angle of 26.7°, compared to the 15° contact angle of the NC control membrane. The NC-GO hybrid membranes were used to separate oligonucleotides that had fewer than 50 nucleotides (nt) from complex solutions. The features of the NC-GO hybrid membranes were tested for extraction periods of 30, 45, and 60 min in three different complex solutions, i.e., an aqueous medium, an α-Minimum Essential Medium (αMEM), and an αMEM supplemented with fetal bovine serum (FBS). The oligonucleotides were desorbed from the surface of the NC-GO hybrid membrane using Tris-HCl buffer with a pH of 8.0. Out of the three media utilized, the best results were achieved after 60 min incubation of the NC-GO membranes in αMEM, as evidenced by the highest fluorescence emission of 294 relative fluorescence units (r.f.u.). This value corresponded to the extraction of approximately 330-370 pg (≈7%) of the total oligo-DNA. This method is an efficient and effortless way to purify short oligonucleotides from complex solutions.

A case of self-improving collodion ichthyosis associated with a rare variant of the ALOX12B gene.

Collodion baby is usually a manifestation of autosomal recessive congenital ichthyosis, a heterogeneous group of congenital hyperkeratotic genodermatoses with highly variable severity and genetic background. Herein, we report a case of self-improving collodion ichthyosis, a rare subtype of autosomal recessive congenital ichthyosis, characterized by an almost-complete spontaneous resolution of symptoms.

Ceramide Analysis in Combination With Genetic Testing May Provide a Precise Diagnosis for Self-Healing Collodion Babies.

Self-healing collodion baby (SHCB), also called "self-improving collodion baby", is a rare mild variant of autosomal recessive congenital ichthyosis and is defined as a collodion baby who shows the nearly complete resolution of scaling within the first 3 months to 1 year of life. However, during the neonatal period, it is not easy to distinguish SHCB from other inflammatory forms of autosomal recessive congenital ichthyosis, such as congenital ichthyosiform erythroderma. Here, we report a case study of two Japanese SHCB patients with compound heterozygous mutations, c.235G>T (p.(Glu79∗))/ c.1189C>T (p.(Arg397Cys)) and c.1295A>G (p.(Tyr432Cys))/ c.1138delG (p.(Asp380Thrfs∗3)), in CYP4F22, which encodes cytochrome P450, family 4, subfamily F, polypeptide 22 (CYP4F22). Immunohistochemically, inflammation with the strong expression of IL-17C, IL-36γ, and TNF-α was seen in the skin at birth. CYP4F22 is an ultra-long-chain FA ω-hydroxylase responsible for ω-O-acylceramide (acylceramide) production. Among the epidermal ceramides, acylceramide is a key lipid in maintaining the epidermal permeability barrier function. We found that the levels of ceramides with ω-hydroxy FAs including acylceramides and the levels of protein-bound ceramides were much lower in stratum corneum samples obtained by tape stripping from SHCB patients than in those from their unaffected parents and individuals without SHCB. Additionally, our cell-based enzyme assay revealed that two mutants, p.(Glu79∗) and p.(Arg397Cys), had no enzyme activity. Our findings suggest that genetic testing coupled with noninvasive ceramide analyses using tape-stripped stratum corneum samples might be useful for the early and precise diagnosis of congenital ichthyoses, including SHCB.

Resistance-Based Lateral Flow Immunosensor with a NFC-Enabled Smartphone for Rapid Diagnosis of Leptospirosis in Clinical Samples.

Leptospirosis is one of the most life-threatening tropical diseases caused by pathogenic Leptospira. To date, a diagnostic device that offers rapid and sensitive detection of leptospires has been still in demand for proper treatment to reduce the mortality rate. Herein, we create a resistance-based lateral flow immunosensor diagnosis device (R-LFI) that integrates near-field communication (NFC) with a portable smartphone for leptospiral detection in clinical samples. A specific monoclonal antibody against the pathogen was coated on a nitrocellulose membrane (NCM) where the test line was collocated. Two electrodes with a sandwich-like configuration were installed employing a conductive double-sided adhesive tape and connected with a NFC smartphone-based detection system. A half-sandwich immunocomplex formation induced high proton conduction, resulting in a considerable decrement in resistive response. The performance of the R-LFI sensor was evaluated using recombinant LipL32 (rLipL32), Leptospira interrogans, and clinical samples. The R-LFI device exhibited linear responses toward rLipL32 protein in phosphate buffer and L. interrogans-spiked healthy human serum samples within the concentration ranging from 1 to 1000 ng mL-1 (limit of detection (LOD): 0.29 ng mL-1) and from 104 to 106 cell mL-1 (LOD: 4.89 × 103 cell mL-1), respectively. Our R-LFI sensor successfully detected L. interrogans-positive clinical samples as confirmed by polymerase chain reaction (PCR). This platform offers high specificity, selectivity, simplicity, miniscule sample volume, and no labeling element requirement. These desirable features make it particularly suitable for countries where medical facilities and resources are limited.

Automated reaction mechanisms and kinetics with the nudged elastic band method-based AMK_Mountain and its description of the preliminary alkaline hydrolysis of nitrocellulose monomer.

To improve the transition state (TS) search capability in complex chemical environments, AMK_Mountain is constructed based on the automated reaction mechanisms and kinetics (AutoMeKin) package. AMK_Mountain does not distinguish the reaction type of the TSs, which is beneficial to obtaining a more comprehensive reaction mechanism. In this study, the first step of the alkaline hydrolysis process of nitrocellulose monomer was adopted as the research object, and 730 possible initial configurations are constructed and 22 TSs pass high-level calculations. Energy difference and interaction region indicator reveal that the first step of alkaline hydrolysis is mainly the combination of nitrogen-containing functional groups at the positions α and ß with hydroxide anions, followed by the formation of nitric acid and the further loss of protons to form nitrate. Overall, in combination with GFN2 -xTB and ORCA, the AMK_Mountain technique provides a reliable method for the location of the TSs in complex environments.

Urea, salts, and Tween 20 influence on adsorption of IgG and Leishmania rNTPDase2 to nitrocellulose.

Lateral flow immunochromatography is a widely used technique for immunological assays. Construction of test and control lines is mostly done by antigen adsorption to nitrocellulose membranes, a process not fully understood. This study aimed to evaluate the influence of urea, salts, and Tween 20, on adsorption. The performance of canine IgG in water and in buffer containing urea and salts (pH 8.3) were compared to observe if the interferents would lead to protein stripping when challenged with increasing concentrations of Tween 20 in the lateral flow buffer. Immobilization of the rLiNTPDase2, an antigen for Canine Leishmaniasis diagnosis, was evaluated and compared to the rLbNTPDase2 by the same method. There were no differences between adsorption coefficients of IgG in water and in buffer, but high salt and urea concentrations seems to stabilize and enhance IgG immobilization. Adsorption performance between canine IgG and rNTPDases had different patterns, but was highly similar between rNTPDases, indicating that protein identity may have an important role. Also, low concentrations of Tween 20 in the flow solution may aid the maintenance of rNTPDase2 on the strips. Our results bring insights about protein adsorption and perspectives about the influence of urea, salts and Tween 20 on this process.

Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China.

BACKGROUND: The existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host's immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed. RESULTS: The truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%. CONCLUSIONS: This study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.

Proof-of-concept for speedy development of rapid and simple at-home method for potential diagnosis of early COVID-19 mutant infections using nanogold and aptamer.

The positive single-stranded nature of COVID-19 mRNA led to the low proof-reading efficacy for its genome authentication. Thus mutant covid-19 strains have been rapidly evolving. Besides Alpha, Beta, Gamma, Delta, and Omicron variants, currently, subvariants of omicron are circulating, including BA.4, BA.5, and BA.2.12.1. Therefore, the speedy development of a rapid, simple, and easier diagnosis method to deal with new mutant covid viral infection is critically important. Many diagnosis methods have been developed for COVID-19 detection such as RT-PCR and antibodies detection. However, the former is time-consuming, laborious, and expensive, and the latter relies on the production of antibodies making it not suitable for the early diagnosis of viral infection. Many lateral-flow methods are available but might not be suitable for detecting the mutants, Here we proved the concept for the speedy development of a simple, rapid, and cost-effective early at-home diagnosis method for mutant Covid-19 infection by combining a new aptamer. The idea is to use the current lateral flow Covid-19 diagnosis system available in the market or to use one existing antibody for the Lateral Flow Nitrocellulose filter. To prove the concept, the DNA aptamer specific to spike proteins (S-proteins) was conjugated to gold nanoparticles and served as a detection probe. An antibody that is specific to spike proteins overexpressed on COVID viral particles was used as a second probe immobilized to the nitrocellulose membrane. The aptamer conjugated nanoparticles were incubated with spike proteins for half an hour and tested for their ability to bind to antibodies anchored on the nitrocellulose membrane. The gold nanoparticles were visualized on the nitrocellulose membrane due to interaction between the antigen (S-protein) with both the aptamer and the antibody. Thus, the detection of viral antigen can be obtained within 2 h, with a cost of less than $5 for the diagnosis reagent. In the future, as long as the mutant of the newly emerged viral surface protein is reported, a peptide or protein corresponding to the mutation can be produced by peptide synthesis or gene cloning within several days. An RNA or DNA aptamer can be generated quickly via SELEX. A gold-labeled aptamer specific to spike proteins (S-proteins) will serve as a detection probe. Any available lateral-flow diagnosis kits with an immobilized antibody that has been available on the market, or simply an antibody that binds COVID-19 virus might be used as a second probe immobilized on the nitrocellulose. The diagnosis method can be carried out by patients at home if a clinical trial verifies the feasibility and specificity of this method.

Elaboration, Characterization and Thermal Decomposition Kinetics of New Nanoenergetic Composite Based on Hydrazine 3-Nitro-1,2,4-triazol-5-one and Nanostructured Cellulose Nitrate.

This research aims to develop new high-energy dense ordinary- and nano-energetic composites based on hydrazine 3-nitro-1,2,4-triazol-5-one (HNTO) and nitrated cellulose and nanostructured nitrocellulose (NC and NMCC). The elaborated energetic formulations (HNTO/NC and HNTO/NMCC) were fully characterized in terms of their chemical compatibility, morphology, thermal stability, and energetic performance. The experimental findings implied that the designed HNTO/NC and HNTO/NMCC formulations have good compatibilities with attractive characteristics such as density greater than 1.780 g/cm3 and impact sensitivity around 6 J. Furthermore, theoretical performance calculations (EXPLO5 V6.04) displayed that the optimal composition of the as-prepared energetic composites yielded excellent specific impulses and detonation velocities, which increased from 205.7 s and 7908 m/s for HNTO/NC to 209.6 s and 8064 m/s for HNTO/NMCC. Moreover, deep insight on the multi-step kinetic behaviors of the as-prepared formulations was provided based on the measured DSC data combined with isoconversional kinetic methods. It is revealed that both energetic composites undergo three consecutive exothermic events with satisfactory activation energies in the range of 139-166 kJ/mol for HNTO/NC and 119-134 kJ/mol for HNTO/NMCC. Overall, this research displayed that the new developed nanoenergetic composite based on nitrated cellulose nanostructure could serve as a promising candidate for practical applications in solid rocket propellants and composite explosives.

Novel Pathogenic Mutation of PNPLA1 Identified in Autosomal Recessive Congenital Ichthyosis: A Case Report.

Autosomal recessive congenital ichthyosis (ARCI) is characterized by being born as collodion babies, hyperkeratosis, and skin scaling. We described a collodion baby at birth with mild ectropion, eclabium, and syndactyly. Whole exome sequencing showed a compound heterozygous variant c.[56C>A], p.(Ser19X) and c.[100G>A], p.(Ala34Thr) in the PNPLA1 gene [NM_001145717; exon 1]. The protein encoded by PNPLA1 acts as a unique transacylase that specifically transfers linoleic acid from triglyceride to ω-hydroxy fatty acid in ceramide, thus giving rise to ω-O-acylceramide, a particular class of sphingolipids that is essential for skin barrier function. The variant was located in the patatin core domain of PNPLA1 and resulted in a truncated protein which could disrupt the function of the protein. This case report highlights a novel compound heterozygous mutation in PNPLA1 identified in a Chinese child.

Simultaneously targeting nitrocellulose and antibody by a dual-headed protein.

Immobilizing antibodies on the nitrocellulose membrane is an important step to increase the sensitivity of the Lateral Flow Test strip for detecting pathogenic antigen. In our research, the fusion protein between nitrocellulose-binding anchor protein 3-Helix - a protein that has a strong affinity to nitrocellulose membrane and protein A - a protein that can bind to the Fc tail of IgG antibody was generated. This fusion protein was expected to help IgG antibodies to be more strongly binding and oriented immobilized onto the nitrocellulose membrane. The recombinant vector pET22b-proA and pET22b-proA-3-Helix coded for protein A and protein A-3-Helix were cloned. These proteins were overexpressed in BL21 and purified by immobilized metal affinity chromatography with purity above 90%. The purified protein was used to evaluate the orientation binding on nitrocellulose membranes by lateral flow challenge. Results showed that protein A-3-Helix binding to nitrocellulose membrane was better than that of protein A. The former protein increased antibody binding and stereochemical immobilizing onto nitrocellulose membrane compared to its protein A counterpart. In summary, we have succeeded in cloning, purifying, and characterizing a dual-head recombinant protein A and protein A-3-Helix. The results show the potential application of protein A-3-Helix in the immobilizing antibody on the test strip.

Three dimensionally printed nitrocellulose-based microfluidic platform for investigating the effect of oxygen gradient on cells.

In this article, we present a novel nitrocellulose-based microfluidic chip with 3-dimensional (3D) printing technology to study the effect of oxygen gradient on cells. Compared with conventional polydimethylsiloxane (PDMS) chips of oxygen gradient for cell cultures that can only rely on fluorescence microscope analysis, this hybrid nitrocellulose-based microfluidic platform can provide a variety of analysis methods for cells, including flow cytometry, western blot and RT-PCR, because the nitrocellulose-based chips with cells can be taken out from the growth chambers of 3D printed microfluidic chip and then used for cell collection or lysis. These advantages allow researchers to acquire more information and data on the basic biochemical and physiological processes of cell life. The effect of oxygen gradient on the zebrafish cells (ZF4) was used as a model to show the performance and application of our platform. Hypoxia caused the increase of intercellular reactive oxygen species (ROS) and accumulation of hypoxia-inducible factor 1α (HIF-1α). Hypoxia stimulated the transcription of hypoxia-responsive genes vascular endothelial growth factor (VEGF) and induced cell cycle arrest of ZF4 cells. The established platform is able to obtain more information from cells in response to different oxygen concentration, which has potential for analyzing the cells under a variety of pathological conditions.

DNA immobilization and detection using DNA binding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNA binding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNA binding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNA binding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNA binding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNA binding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.