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A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray
摘要： A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.
关键词： gene expression,enzyme-free,fluorescence microscopy,mRNA detection,microarray
更新于2025-11-21 11:24:58
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Pre-conditioning with Remote Photobiomodulation Modulates the Brain Transcriptome and Protects Against MPTP Insult in Mice
摘要： Transcranial photobiomodulation (PBM), which involves the application of low-intensity red to near-infrared light (600-1100nm) to the head, provides neuroprotection in animal models of various neurodegenerative diseases. However, the absorption of light energy by the human scalp and skull may limit the utility of transcranial PBM in clinical contexts. We have previously shown that targeting light at peripheral tissues (i.e. “remote PBM”) also provides protection of the brain in an MPTP mouse model of Parkinson’s disease, suggesting remote PBM might be a viable alternative strategy for overcoming penetration issues associated with transcranial PBM. This present study aimed to determine an effective pre-conditioning regimen of remote PBM for inducing neuroprotection and elucidate the molecular mechanisms by which remote PBM enhances the resilience of brain tissue. Balb/c mice were irradiated with 670nm light (4J/cm2 per day) targeting dorsum and hindlimbs for 2, 5 or 10 days, followed by injection of the parkinsonian neurotoxin MPTP (50mg/kg) over two consecutive days. Despite no direct irradiation of the head, 10 days of pre-conditioning with remote PBM significantly attenuated MPTP-induced loss of midbrain tyrosine hydroxylase-positive dopaminergic cells and mitigated the increase in FOS-positive neurons in the caudate-putamen complex. Interrogation of the midbrain transcriptome by RNA microarray and pathway enrichment analysis suggested upregulation of cell signaling and migration (including CXCR4+ stem cell and adipocytokine signaling), oxidative stress response pathways and modulation of the blood-brain barrier following remote PBM. These findings establish remote PBM preconditioning as a viable neuroprotective intervention and provide insights into the mechanisms underlying this phenomenon.
关键词： MPTP,microarray,Parkinson’s disease,mouse model,neuroprotection,photobiomodulation
更新于2025-09-23 15:23:52
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The Impact of Photobleaching on Microarray Analysis
摘要： DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner’s laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube’s voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results.
关键词： photobleaching,microarray,cyanine dye,bioanalytics,fluorophore,bioinformatics,DNA
更新于2025-09-23 15:22:29
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Development of a Protein Microarray Chip with Enhanced Fluorescence for Identification of Semen and Vaginal Fluid
摘要： The detection of body ?uids has been used to identify a suspect and build a criminal case. As the amount of evidence collected at a crime site is limited, a multiplex identi?cation system for body ?uids using a small amount of sample is required. In this study, we proposed a multiplex detection platform using an Ag vertical nanorod metal enhanced ?uorescence (MEF) substrate for semen and vaginal ?uid (VF), which are important evidence in cases of sexual crime. The Ag nanorod MEF substrate with a length of 500 nm was fabricated by glancing angle deposition, and amino functionalization was conducted to improve binding ability. The effect of incubation time was analyzed, and an incubation time of 60 min was selected, at which the ?uorescence signal was saturated. To assess the performance of the developed identi?cation chip, the identi?cation of semen and VF was carried out. The developed sensor could selectively identify semen and VF without any cross-reactivity. The limit of detection of the fabricated microarray chip was 10 times better than the commercially available rapid stain identi?cation (RSID) Semen kit.
关键词： metal-enhanced ?uorescence,glancing angle deposition,microarray analysis,semen,vaginal ?uid,Ag nanorod,body ?uid identi?cation
更新于2025-09-23 15:21:21
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A versatile fluorometric in-situ hybridization method for the quantitation of hairpin conformations in DNA self-assembled monolayers
摘要： As the performance of hairpin DNA (hpDNA)-based biosensors is highly dependent on the yield of stem-loop (hairpin) conformations, we report herein a versatile fluorometric in-situ hybridization protocol for examining hpDNA self-assembled monolayers (SAMs) on popularly used biochip substrates. Specifically, the ratio of fluorescence (FL) intensities of hpDNA SAMs (in an array format) before and after hybridization was adapted as the key parameter for performing such a determination. Upon confirming the existence of mixed and tunable DNA conformations in binary deposition solutions and the efficient hybridization of the hairpin strands with target DNA via gel electrophoresis assays, we have tested the fluorometric protocol for determining the coverages of hpDNA in hpDNA/ssDNA SAMs prepared on gold; its accuracy was validated by Exonuclease I (Exo I)-assisted electrochemical quantitation. To further confirm its versatility, this FL protocol was adapted for quantifying hairpin conformations formed on glass and polycarbonate (PC) substrates. The molar ratios of surface-tethered hairpin conformations on the three different substrates were all found to be proportional to but less than that in the binary deposition solutions, and dependent on the substrate morphology. The findings reported herein are beneficial to the construction of highly efficient DNA hairpin-based sensing surfaces, which essentially facilitates the creation of hpDNA-based biosensors with optimal detection performance.
关键词： In-situ hybridization,conformation evaluation,DNA biosensor/biochip,fluorescence microarray,Hairpin DNA
更新于2025-09-23 15:19:57
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A Low‐Cost Laser‐Based Nano‐3D Polymer Printer for Rapid Surface Patterning and Chemical Synthesis of Peptide and Glycan Microarrays
摘要： A low-cost laser-based printing setup is presented, which allows for the spot-wise patterning of surfaces with defined polymer nanolayers. These nanolayer spots serve as a “solid solvent,” embedding different chemicals, chemical building blocks, materials, or precursors and can be stacked on top of each other. By melting the spot pattern, the polymer-embedded molecules are released for chemical reaction. This enables researchers to quickly pattern a surface with different molecules and materials, mixing them directly on the surface for high-throughput chemical synthesis to generate and screen diverse microarray libraries. In contrast to expensive ink-jet or contact printing, this approach does not require premixing of inks, which enables in situ combinatorial mixing. Easy access and versatility of this patterning approach are shown by generating microarrays of various biomolecules, such as glycans for the first time, to screen interactions of antibodies and lectins. In addition, a layer-by-layer solid-phase synthesis of peptides directly on the microarray is presented. Amino acid–containing nanolayers are repeatedly laser-transferred and reacted with the functionalized acceptor surface in defined patterns. This simple system enables a reproducible array production, down to spot-to-spot distances of 100 μm, and offers a flexible and cheap alternative to expensive spotting robot technology.
关键词： microarray,open source,laser-induced forward transfer,combinatorial synthesis,solid phase synthesis
更新于2025-09-19 17:13:59
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Label-free bacteria quantification in blood plasma by a bioprinted microarray based interferometric point-of-care device
摘要： Existing clinical methods for bacteria detection lack in speed, sensitivity and importantly in Point-of-Care (PoC) applicability. Thus, finding ways to push the sensitivity of clinical PoC biosensing technologies is crucial. Aiming that, we here report a portable PoC device based on Lens-free Interferometric Microscopy (LIM). The device employs high performance nanoplasmonics and custom bioprinted microarrays and is capable of direct label-free bacteria (E. coli) quantification. With only one-step sample handling we offer a sample?to?data turnaround time of 40 minutes. Our technology features detection sensitivity of a single bacterial cell both in buffer and diluted blood plasma and is intrinsically limited by the number of cells present in the detection volume. When employed in a hospital setting, the device has enabled accurate categorization of sepsis patients (infectious SIRS) from control groups (healthy individuals and non-infectious SIRS patients) without false positives/negatives. User-friendly on-site bacterial clinical diagnosis can thus become a reality.
关键词： microarray,label-free detection,plasma samples,nanoplasmonics,sepsis,bacteria
更新于2025-09-09 09:28:46
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SERS-Based Quantification of PSMA in Tissue Microarrays Allows Effective Stratification of Patients with Prostate Cancer
摘要： Prostate specific membrane antigen (PSMA), a type II membrane protein, is an attractive biomarker that has been validated clinically for the diagnosis of prostate cancer. In this study, we developed surface-enhanced Raman scattering (SERS) nanoprobes for PSMA detection and quantification at the single-cell level on prostate cancer cells. The cells were targeted employing SERS nanoprobes that consisted of gold nanostars functionalized with PSMA aptamer molecules. We were able to quantify picomolar concentrations of soluble PSMA protein and used the resulting calibration curve to estimate the expression of PSMA on the surface of the prostate cancer cell, LNCaP, at the single-cell level. Importantly, we employed these SERS tags to stratify prostate cancer patients by assessing PSMA expression in tissues contained in a prostate tissue microarray. The stratification results clearly correlated PSMA expression to recommended therapy groups, rendering the described method as an effective tool to aid in designing personalized therapeutic protocols. Benchmarking detection sensitivity against immunofluorescence staining and comparing stratification results obtained with the two methods allowed us to validate our novel approach against standard practices. On the basis of these results, we confirm the validity of PSMA as an effective biomarker for prostate cancer patient evaluation and propose SERS-based diagnostic techniques as integrative methods for the assessment of disease stage and the identification of effective therapeutic protocols.
关键词： aptamer,tissue microarray,surface-enhanced Raman scattering,PSMA,Prostate specific membrane antigen,SERS,nanoprobes,prostate cancer,biomarker,gold nanostars
更新于2025-09-04 15:30:14
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Characterization of a DNA Aptamer for Ovarian Cancer Clinical Tissue Recognition and in Vivo Imaging
摘要： Backgrounds/Aims: Ovarian cancer is the most lethal gynaecologic malignancy and is difficult to detect early. The inefficient early diagnosis of ovarian cancer is the main contributor to its high mortality rate. Aptamers, as chemical antibodies, are single-stranded DNA or RNA oligonucleotides that target cells or molecules with high affinity. Methods: Binding ability of R13 was measured by flow cytometry analysis. Stability of R13 was tested in blood serum of an ovarian cancer patient. Internalization of R13 was verified by confocal microscope imaging. 80 cases ovarian cancer tissues, 10 cases normal ovary tissues in a microarray and 6 fallopian tube tissues were prepared for this study. R13’s target ability was further confirmed in vivo tumor models in NOD/SCID mice. Results: In this study, we found aptamer R13 bound to ovarian cancer cells with dissociation constants in the nanomolar range. Moreover, these results were further confirmed by tissue imaging. Next we demonstrated that the targets of R13 are membrane proteins and that its internalization occurs in a caveolae-mediated and clathrin-mediated manner. The target function of R13 was determined by imaging A2780 tumours in mouse models. Conclusion: These findings suggest that R13 is a promising novel tool to diagnose and deliver drugs to treat ovarian cancer.
关键词： Endocytosis,DNA aptamer,Membrane proteins,Ovarian cancer,Nude mouse,Tissue microarray
更新于2025-09-04 15:30:14
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Improvement in the amine glass platform by bubbling method for a DNA microarray
摘要： A glass platform with high sensitivity for sexually transmitted diseases microarray is described here. An amino-silane-based self-assembled monolayer was coated on the surface of a glass platform using a novel bubbling method. The optimized surface of the glass platform had highly uniform surface modifications using this method, as well as improved hybridization properties with capture probes in the DNA microarray. On the basis of these results, the improved glass platform serves as a highly reliable and optimal material for the DNA microarray. Moreover, in this study, we demonstrated that our glass platform, manufactured by utilizing the bubbling method, had higher uniformity, shorter processing time, lower background signal, and higher spot signal than the platforms manufactured by the general dipping method. The DNA microarray manufactured with a glass platform prepared using bubbling method can be used as a clinical diagnostic tool.
关键词： glass platform,bubbling method,self-assambled monolayer,DNA microarray
更新于2025-09-04 15:30:14