SERS sensing for cancer biomarker: Approaches and directions.

These days, cancer is thought to be more than just one illness, with several complex subtypes that require different screening approaches. These subtypes can be distinguished by the distinct markings left by metabolites, proteins, miRNA, and DNA. Personalized illness management may be possible if cancer is categorized according to its biomarkers. In order to stop cancer from spreading and posing a significant risk to patient survival, early detection and prompt treatment are essential. Traditional cancer screening techniques are tedious, time-consuming, and require expert personnel for analysis. This has led scientists to reevaluate screening methodologies and make use of emerging technologies to achieve better results. Using time and money saving techniques, these methodologies integrate the procedures from sample preparation to detection in small devices with high accuracy and sensitivity. With its proven potential for biomedical use, surface-enhanced Raman scattering (SERS) has been widely used in biosensing applications, particularly in biomarker identification. Consideration was given especially to the potential of SERS as a portable clinical diagnostic tool. The approaches to SERS-based sensing technologies for both invasive and non-invasive samples are reviewed in this article, along with sample preparation techniques and obstacles. Aside from these significant constraints in the detection approach and techniques, the review also takes into account the complexity of biological fluids, the availability of biomarkers, and their sensitivity and selectivity, which are generally lowered. Massive ways to maintain sensing capabilities in clinical samples are being developed recently to get over this restriction. SERS is known to be a reliable diagnostic method for treatment judgments. Nonetheless, there is still room for advancement in terms of portability, creation of diagnostic apps, and interdisciplinary AI-based applications. Therefore, we will outline the current state of technological maturity for SERS-based cancer biomarker detection in this article. The review will meet the demand for reviewing various sample types (invasive and non-invasive) of cancer biomarkers and their detection using SERS. It will also shed light on the growing body of research on portable methods for clinical application and quick cancer detection.

Optimization of human umbilical cord blood-derived mesenchymal stem cell isolation and culture methods in serum- and xeno-free conditions.

BACKGROUND: Although umbilical cord blood (UCB) is identified as a source of mesenchymal stem cells (MSCs) with various advantages, the success in cell isolation is volatile. Therefore, it is necessary to optimize methods of cord blood-derived MSC (UCB-MSC) isolation and culture. In this study, we evaluated the efficiency of UCB-MSC isolation and expansion using different commercially available serum- and xeno-free media and investigated the capacity of autologous serum and plasma as a supplement to support cell proliferation. Additionally, we defined the presence of multilineage-differentiating stress-enduring (Muse) cells in the UCB-MSC population. Functions of UCB-MSC in in vitro angiogenesis processes and anti-cancer were also verified. METHODS: Mononuclear cells were isolated using density gradient separation and cultured in four commercial media kits, as well as four surface coating solutions. UCB-MSCs were characterized and tested on tube formation assay, and co-cultured with SK-MEL cells in a transwell system. RESULTS: The results showed that only StemMACS™ MSC Expansion Media is more appropriate to isolate and culture UCB-MSCs. The cells exhibited a high cell proliferation rate, CFU forming capability, MSC surface marker expression, trilineage differentiate potential, and chromosome stability. In addition, the culture conditions with autologous serum coating and autologous plasma supplement enhanced cell growth and colony forming. This cell population contained Muse cells at rate of 0.3%. Moreover, UCB-MSCs could induce the tube formation of human umbilical vein endothelial cells and inhibit more than 50% of SK-MEL cell growth. CONCLUSIONS: UCB-MSCs could be high-yield isolated and expanded under serum- and xeno-free conditions by using the StemMACS™ MSC Expansion Media kit. Autologous serum coating and plasma supplement enhanced cell proliferation. These UCB-MSCs had effected the tube formation process and an anti-cancer impact.

Factors Affecting Human Umbilical Cord Blood Quality Before Cryopreservation: The Importance of Birth Weight and Gestational Age.

Background: Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells and is useful for the treatment of blood diseases. The cost of UCB storage is high; thus, it is necessary to evaluate the quality of UCB before collection and cryopreservation. Aim: This study aimed to determine the maternal and neonatal factors that influence UCB before selection for cryopreservation. Materials and Methods: The analysis included 403 processed UCB units. The effects of maternal characteristics including maternal age and delivery method and neonatal factors such as birth weight, gestation duration, and sex on UCB quality were determined based on the collected blood volume, total nucleated cell (TNC) count, and CD34+ cell count. Results: The neonatal birth weight influenced the collected blood volume, TNC count, and CD34+ cell count. Neonates with higher birth weights produced better quality UCB units because of increased collected blood volumes, TNC counts, and CD34+ cell counts. However, an increase in the gestational age from 35 to 41 weeks led to decreases in the collected blood volume and CD34+ cell count. Conclusion: These data may be useful for determining the optimal cord blood units for collection and cryopreservation and for advising pregnant women using private banking services.

A Sensitive, Specific and Simple Loop Mediated Isothermal Amplification Method for Rapid Detection of Campylobacter spp. in Broiler Production.

Campylobacteriosis is one of the most common foodborne diseases worldwide. Two Campylobacter species - C. jejuni and C. coli in poultry and poultry products are considered to be the main source of human campylobacteriosis. Therefore, studying Campylobacter status in poultry flocks is needed to prevent transmission of disease and reduce human risk, health cost, and economic losses. In this study, we adapted and used a Loop-Mediated Isothermal Amplification (LAMP) assay for specific, sensitive, simple and cost-effective rapid detection of C. jejuni and C. coli in the poultry production chain. Amplified LAMP products were detected using a small, low-cost portable commercial blue LED transilluminator and a direct visual detection strategy was demonstrated. By using optimized conditions for amplification a limit of detection (LOD) of 50 CFU/ml was achieved for testing of C. jejuni and C. coli in spiked chicken feces without enrichment. The method took 60-70 min from receiving the samples to the final results (including 30 min for amplification). The optimized LAMP showed a relative accuracy of 98.4%, a specificity of 97.9%, and a sensitivity of 100% in comparison to real-time PCR method. Cohen's kappa index also showed an excellent agreement (0.94) between the two methods. The results showed that the method is specific, sensitive and is suitable to develop for rapid detection of Campylobacter spp. at poultry production.

Classification of Multiple DNA Dyes Based on Inhibition Effects on Real-Time Loop-Mediated Isothermal Amplification (LAMP): Prospect for Point of Care Setting.

LAMP has received great interest and is widely utilized in life sciences for nucleic acid analysis. To monitor a real-time LAMP assay, a fluorescence DNA dye is an indispensable component and therefore the selection of a suitable dye for real-time LAMP is a need. To aid this selection, we investigated the inhibition effects of twenty-three DNA dyes on real-time LAMP. Threshold time (Tt) values of each real-time LAMP were determined and used as an indicator of the inhibition effect. Based on the inhibition effects, the dyes were classified into four groups: (1) non-inhibition effect, (2) medium inhibition effect, (3) high inhibition effect, and (4) very high inhibition effect. The signal to noise ratio (SNR) and the limit of detection (LOD) of the dyes in groups 1, 2, and 3 were further investigated, and possible inhibition mechanisms of the DNA dyes on the real-time LAMP are suggested and discussed. Furthermore, a comparison of SYTO 9 in different LAMP reactions and different systems is presented. Of the 23 dyes tested, SYTO 9, SYTO 82, SYTO 16, SYTO 13, and Miami Yellow were the best dyes with no inhibitory effect, low LOD and high SNR in the real-time LAMP reactions. The present classification of the dyes will simplify the selection of fluorescence dye for real-time LAMP assays in point of care setting.

Protein adaptors assemble functional proteins on DNA scaffolds.

DNA is an attractive molecular building block to construct nanoscale structures for a variety of applications. In addition to their structure and function, modification the DNA nanostructures by other molecules opens almost unlimited possibilities for producing functional DNA-based architectures. Among the molecules to functionalize DNA nanostructures, proteins are one of the most attractive candidates due to their vast functional variations. DNA nanostructures loaded with various types of proteins hold promise for applications in the life and material sciences. When loading proteins of interest on DNA nanostructures, the nanostructures by themselves act as scaffolds to specifically control the location and number of protein molecules. The methods to arrange proteins of interest on DNA scaffolds at high yields while retaining their activity are still the most demanding task in constructing usable protein-modified DNA nanostructures. Here, we provide an overview of the existing methods applied for assembling proteins of interest on DNA scaffolds. The assembling methods were categorized into two main classes, noncovalent and covalent conjugation, with both showing pros and cons. The recent advance of DNA-binding adaptor mediated assembly of proteins on the DNA scaffolds is highlighted and discussed in connection with the future perspectives of protein assembled DNA nanoarchitectures.

Solid Phase PCR on 3D Microstructure ArrayChip for Pathogen Detection Application.

Advanced free angle photolithography (FAPL) is presented for making 3D supercritical angle fluorescence (SAF) microstructures and transfer them on to polymeric chips using injection molding technique for low-cost microfluidic devices embedded with optical sensing structures. A solid phase polymerase chain reaction (SP-PCR) is used as model technique, which allows rapid and sensitive detection of pathogen DNA on-chip. This article presents the detailed fabrication of SAF structure and SP-PCR application on SAF structure for pathogen detection. This protocol of developing SAF structures using the FAPL process, increases the number of SAF per mm2. FAPL was performed via a motorized stage to control the angle of incidence and to achieve the desired bucket-shapes (dimensions of 50 µm to 150 µm with a slope) required for the 3D optical sensing. Due to the unique properties of SAF structures, it enhances the fluorescent signal by 46 times. Increasing the number of SAF structures and reducing the size resulted in reduction of sample volume required per test along with improvement in the limit of detection (LOD) due to a smaller size. This article also presents the experimental details of SP-PCR using DNA oligos bound to the SAF structures for on-chip pathogen detection and a comparison between different sizes of SAF structures. The direct on-chip SP-PCR paves the path for the application of this technique in point-of-care devices.

MicroRNA amplification and detection technologies: opportunities and challenges for point of care diagnostics.

The volume of point of care (POC) testing continues to grow steadily due to the increased availability of easy-to-use devices, thus making it possible to deliver less costly care closer to the patient site in a shorter time relative to the central laboratory services. A novel class of molecules called microRNAs have recently gained attention in healthcare management for their potential as biomarkers for human diseases. The increasing interest of miRNAs in clinical practice has led to an unmet need for assays that can rapidly and accurately measure miRNAs at the POC. However, the most widely used methods for analyzing miRNAs, including Northern blot-based platforms, in situ hybridization, reverse transcription qPCR, microarray, and next-generation sequencing, are still far from being used as ideal POC diagnostic tools, due to considerable time, expertize required for sample preparation, and in terms of miniaturizations making them suitable platforms for centralized labs. In this review, we highlight various existing and upcoming technologies for miRNA amplification and detection with a particular emphasis on the POC testing industries. The review summarizes different miRNA targets and signals amplification-based assays, from conventional methods to alternative technologies, such as isothermal amplification, paper-based, oligonucleotide-templated reaction, nanobead-based, electrochemical signaling- based, and microfluidic chip-based strategies. Based on critical analysis of these technologies, the possibilities and feasibilities for further development of POC testing for miRNA diagnostics are addressed and discussed.

Microfluidic devices for sample preparation and rapid detection of foodborne pathogens.

Rapid detection of foodborne pathogens at an early stage is imperative for preventing the outbreak of foodborne diseases, known as serious threats to human health. Conventional bacterial culturing methods for foodborne pathogen detection are time consuming, laborious, and with poor pathogen diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices for online monitoring of pathogens with high accuracy and sensitivity in a time-saving and cost effective manner. Lab on chip is a blooming area in diagnosis, which exploits different mechanical and biological techniques to detect very low concentrations of pathogens in food samples. This is achieved through streamlining the sample handling and concentrating procedures, which will subsequently reduce human errors and enhance the accuracy of the sensing methods. Integration of sample preparation techniques into these devices can effectively minimize the impact of complex food matrix on pathogen diagnosis and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods and food production line.

Spatially Organized Enzymes Drive Cofactor-Coupled Cascade Reactions.

We report the construction of an artificial enzyme cascade based on the xylose metabolic pathway. Two enzymes, xylose reductase and xylitol dehydrogenase, were assembled at specific locations on DNA origami by using DNA-binding protein adaptors with systematic variations in the interenzyme distances and defined numbers of enzyme molecules. The reaction system, which localized the two enzymes in close proximity to facilitate transport of reaction intermediates, resulted in significantly higher yields of the conversion of xylose into xylulose through the intermediate xylitol with recycling of the cofactor NADH. Analysis of the initial reaction rate, regenerated amount of NADH, and simulation of the intermediates' diffusion indicated that the intermediates diffused to the second enzyme by Brownian motion. The efficiency of the cascade reaction with the bimolecular transport of xylitol and NAD(+) likely depends more on the interenzyme distance than that of the cascade reaction with unimolecular transport between two enzymes.

A modular zinc finger adaptor accelerates the covalent linkage of proteins at specific locations on DNA nanoscaffolds.

A modular adaptor consisting of a sequence-specific DNA binding zinc finger protein and a self-ligating protein-tag was developed to expedite efficient formation of a covalent linkage between an individual protein molecule and the programmed address modified with a tag-substrate on the DNA nanostructure.

A protein adaptor to locate a functional protein dimer on molecular switchboard.

The addressable DNA nanostructures offer ideal platforms to construct organized assemblies of multiple protein molecules. Sequence-specific DNA binding proteins that target defined sites on DNA nanostructures would act as orthogonal adaptors to carry individual protein molecules to the programmed addresses. We have recently developed a protein-based adaptor by utilizing the sequence-specific DNA binding zinc finger protein to locate a monomeric protein of interest at specific positions on DNA origami, which serves as a molecular switchboard. We herein report a new adaptor to locate a protein dimer on the DNA origami scaffold based on a homodimeric basic-leucine zipper protein GCN4. Specific binding of GCN4 to programmed addresses on DNA origami and orthogonal targeting by GCN4- and zinc finger protein-based adaptors to the respective addresses on DNA origami were confirmed by gel electrophoretic and AFM analyses. Furthermore, a GCN4-fused homodimeric enzyme showed even higher activity than the wild type enzyme, and exhibited avid reactivity when assembled at the specific site of DNA origami. Thus, GCN4 serves as an ideal adaptor to locate homodimeric proteins in the functional form on DNA origami.