Rapid photothermal assay for ultrasensitive point-of-care detection of tumor markers based on a filter membrane.

An ultrasensitive photothermal assay was designed for point-of-care testing (POCT) of tumor markers based on a filter membrane. Firstly, Cu2-xSe was successfully encapsulated in liposome spheres with biotin on the surface and connected to carcinoembryonic antigen (CEA) aptamer with 3'end modified biotin by streptavidin. Secondly, the CEA antibody was successfully modified on the surface of the nitrocellulose membrane through simple incubation. Finally, the assay process was completed using a disposable syringe, and the temperature was recorded using a handheld infrared temperature detector. In the range 0-50 ng mL-1, the temperature change of the nitrocellulose membrane has a strong linear relationship with CEA concentration, and the detection limit is 0.097 ng mL-1. It is worth noting that the entire testing process can be easily performed in 10 min, much shorter than traditional clinical methods. In addition, this method was successfully applied to the quantitative determination of CEA levels in human serum samples with a recovery of 96.2-103.3%. This rapid assay can be performed by "one suction and one push" through a disposable syringe, which is simple to operate, and the excellent sensitivity reveals the great potential of the proposed strategy in the POCT of tumor biomarkers.

Laser-assembled conductive 3D nanozyme film-based nitrocellulose sensor for real-time detection of H2O2 released from cancer cells.

In this work, a nanostructured conductive film possessing nanozyme features was straightforwardly produced via laser-assembling and integrated into complete nitrocellulose sensors; the cellulosic substrate allows to host live cells, while the nanostructured film nanozyme activity ensures the enzyme-free real-time detection of hydrogen peroxide (H2O2) released by the sames. In detail, a highly exfoliated reduced graphene oxide 3D film decorated with naked platinum nanocubes was produced using a CO2-laser plotter via the simultaneous reduction and patterning of graphene oxide and platinum cations; the nanostructured film was integrated into a nitrocellulose substrate and the complete sensor was manufactured using an affordable semi-automatic printing approach. The linear range for the direct H2O2 determination was 0.5-80 µM (R2 = 0.9943), with a limit of detection of 0.2 µM. Live cell measurements were achieved by placing the sensor in the culture medium, ensuring their adhesion on the sensors' surface; two cell lines were used as non-tumorigenic (Vero cells) and tumorigenic (SKBR3 cells) models, respectively. Real-time detection of H2O2 released by cells upon stimulation with phorbol ester was carried out; the nitrocellulose sensor returned on-site and real-time quantitative information on the H2O2 released proving useful sensitivity and selectivity, allowing to distinguish tumorigenic cells. The proposed strategy allows low-cost in-series semi-automatic production of paper-based point-of-care devices using simple benchtop instrumentation, paving the way for the easy and affordable monitoring of the cytopathology state of cancer cells.

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.

Nitrocellulose/acrylic resin coated screen-printed carbon electrode to construct a capacitive immunosensor for anti-BSA.

The capacitive immunosensor, known for its label-free simplicity, has great potential for point-of-care diagnostics. However, the interaction between insulation and recognition layers on the sensing electrode greatly affects its performance. This study introduces a pioneering dual-layer strategy, implementing a novel combination of acrylic resin (AR) and nitrocellulose (NC) coatings on screen-printed carbon electrodes (SPCEs). This innovative approach not only enhances the dielectric properties of the capacitive sensor but also streamlines the immobilization of recognizing elements. Particularly noteworthy is the superior reliability and insulation offered by the AR coating, surpassing the limitations of traditional self-assembled monolayer (SAM) modifications. This dual-layer methodology establishes a robust foundation for constructing capacitive sensors optimized specifically for liquid medium-based biosensing applications. The NC coating in this study represents a breakthrough in effectively immobilizing BSA, unraveling the capacitive response intricately linked to the quantity of adsorbed recognizing elements. The results underscore the prowess of the proposed immunosensor, showcasing a meticulously defined linear calibration curve for anti-BSA (ranging from 0 to 25 µg/ml). Additionally, specific interactions with anti-HAS and anti-TNF-α further validate the versatility and efficacy of the developed immunosensor. This work presents a streamlined and highly efficient protocol for developing label-free immunosensors for antibody determination and introduces a paradigm shift by utilizing readily available electrodes and sensing systems. The findings are poised to catalyze a significant acceleration in the advancement of biosensor technology, opening new avenues for innovative applications in point-of-care diagnostics.

Reduced graphene oxide electrodes meet lateral flow assays: A promising path to advanced point-of-care diagnostics.

Research in electrochemical detection in lateral flow assays (LFAs) has gained significant momentum in recent years. The primary impetus for this surge in interest is the pursuit of achieving lower limits of detection, especially given that LFAs are the most widely employed point-of-care biosensors. Conventionally, the strategy for merging electrochemistry and LFAs has centered on the superposition of screen-printed electrodes onto nitrocellulose substrates during LFA fabrication. Nevertheless, this approach poses substantial limitations regarding scalability. In response, we have developed a novel method for the complete integration of reduced graphene oxide (rGO) electrodes into LFA strips. We employed a CO2 laser to concurrently reduce graphene oxide and pattern nitrocellulose, exposing its backing to create connection sites impervious to sample leakage. Subsequently, rGO and nitrocellulose were juxtaposed and introduced into a roll-to-roll system using a wax printer. The exerted pressure facilitated the transfer of rGO onto the nitrocellulose. We systematically evaluated several electrochemical strategies to harness the synergy between rGO and LFAs. While certain challenges persist, our rGO transfer technology presents compelling potential for setting a new standard in electrochemical LFA fabrication.

Green manufacturing of a hypoxanthine enzyme sensor for fish freshness based on modified nitrocellulose surface with chito-oligosaccharide.

Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 µM; the linear range is 0.05-0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness.

Non-invasive iron deficiency diagnosis: a saliva-based approach using capillary flow microfluidics.

Iron deficiency anemia (IDA) is a condition characterized by lower-than-average iron (Fe) levels in the body, affecting a substantial number of young children and pregnant women globally. Existing diagnostic methods for IDA rely on invasive analysis of stored Fe in ferritin from blood samples, posing challenges, especially for toddlers and young children. To address this issue, saliva has been proposed as a non-invasive sample matrix for IDA diagnosis. However, conventional Fe analysis techniques often necessitate complex and costly instrumentation. This study presents the first non-invasive, saliva-based preliminary screening test for IDA using a nitrocellulose lateral flow system. In this study, we introduce a novel approach using the ferroin reaction with bathophenanthroline (Bphen) and ferrous (Fe2+) ions to quantify Fe levels in saliva. Our methodology involves a capillary flow-driven microfluidic device integrated into a lateral flow system utilizing nitrocellulose membranes. Here, we present the first instance of saliva on a nitrocellulose substrate to detect salivary Fe levels. The optimized system yielded a linear response over the 1-200 ppm range in buffer solution, with a limit of detection (LoD) of 5.6 ppm. Furthermore, the system demonstrated a linear response in pooled saliva samples across the 1-1000 ppm range, with a LoD of 55.1 ppm. These results underscore the potential of our capillary flow-driven microfluidic device as a viable non-invasive diagnostic tool for IDA, particularly in remote and resource-limited settings.

Autosomal recessive ALOX12B gene and consecutive collodion baby.

Autosomal recessive congenital ichthyosis is a type of inherited ichthyosis which is a rare cluster of genetic disorders leading to defective keratinisation. The combined prevalence for lamellar ichthyosis and congenital ichthyosiform erythroderma is almost 1 per 200 000-300 000 people. Among all the mutations in this gene, missense and frameshift mutations are most common which account for 80% of the cases. Our patient had a mutation in R-type arachidonate 12-lipoxygenase gene (ALOX12B, OMIM*603741).

Solid Phase Oligo-DNA Extraction from Complex Medium Using an Aminated Graphene/Nitrocellulose Membrane Hybrid.

A hybrid material, consisting of commercially available nitrocellulose (NC) membrane non-covalently modified with amino-polyethylene glycol functionalized reduced graphene oxide (NH2-PEG-rGO) nanoparticles, was successfully synthesized for oligonucleotide extraction. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the modification of the NC membrane, revealing characteristic peaks of both compounds, i.e., NC and NH2-PEG-rGO. Scanning Electron Microscopy (SEM) exhibited morphological changes in the NC/NH2-PEG-rGO hybrid membrane, marked by the introduction of NH2-PEG-rGO particles, resulting in a distinctly smothered surface compared to the porous surface of the NC control membrane. Wettability assays revealed hydrophobic behavior for the NC/NH2-PEG-rGO hybrid membrane, with a water contact angle exceeding 90°, contrasting with the hydrophilic behavior characterized by a 16.7° contact angle in the NC membrane. The performance of the NC/NH2-PEG-rGO hybrid membrane was evaluated for the extraction of ssDNA with fewer than 50 nucleotides from solutions containing various ionic species (MnCl2, MgCl2, and MnCl2/MgCl2). The NC/NH2-PEG-rGO hybrid membrane exhibited optimal performance when incubated in MgCl2, presenting the highest fluorescence emission at 525 relative fluorescence units (r.f.u.). This corresponds to the extraction of approximately 610 pg (≈13%) of the total oligo-DNA, underscoring the efficacy of the pristine material, which extracts 286 pg (≈6%) of oligo-DNA in complex solutions.

Integration of a hamper pad on test strips for improved sensitivity of carbendazim detection.

Lateral flow immunoassays (LFIAs) are widely used to determine carbendazim (CBZ) residues in food products due to their advantages of low cost, ease and rapid use, on-site detection capability. However, conventional LFIAs have low detection sensitivity. Although improvements have been made to increase the sensitivity, it is not sufficient. Here, a hamper pad, polyvinyl alcohol coated on a nitrocellulose membrane, was integrated to enhance the sensitivity of LFIA for CBZ detection. The hamper pad was inserted between the conjugated and nitrocellulose pads to delay the flow rate, thereby increasing the possibility of the antibody and target analyte binding. This platform exhibited a fourfold sensitivity increase in CBZ detection compared with the conventional LFIA, and its limit of detection was 1.6 ng/mL. In addition, a single-step operation was successfully applied to detect CBZ in rice (white rice, brown rice, sticky rice, and paddy) and soybean samples, with acceptable recoveries of 93.6%-120.0%. This novel device was compared to the standard high-performance liquid chromatography method, which shows high accuracy with a Kappa coefficient of 0.91. Therefore, improved sensitivity with a rapid, simple, and inexpensive device could facilitate the detection of CBZ residues in agricultural products for on-field screening and improved user-friendliness.

NFC-enabled potentiostat and nitrocellulose-based metal electrodes for electrochemical lateral flow assay.

Rapid detection of pathogens at the point-of-need is crucial for preventing the spread of human, animal and plant diseases which can have devastating consequences both on the lives and livelihood of billions of people. Colorimetric, lateral flow assays consisting of a nitrocellulose membrane, are the preferred format today for low-cost on-site detection of pathogens. This assay format has, however, historically suffered from poor analytical performance and is not compatible with digital technologies. In this work, we report the development of a new class of digital diagnostics platform for precision point-of-need testing. This new versatile platform consists of two important innovations: i) A wireless and batteryless, microcontroller-based, low-cost Near Field Communication (NFC)-enabled potentiostat that brings high performance electroanalytical techniques (cyclic voltammetry, chronoamperometry, square wave voltammetry) to the field. The NFC-potentiostat can be operated with a mobile app by minimally trained users; ii) A new approach for producing nitrocellulose membranes with integrated electrodes that facilitate high performance electrochemical detection at the point-of-need. We produced an integrated system housed in a 3D-printed phone case and demonstrated its use for the detection of Maize Mosaic Virus (MMV), a plant pathogen, as a proof-of-concept application.

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina.

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

Immunoblot Analysis from Single Cells Using Milo™ Single-Cell Western Platform.

In this new era of precision medicine, characterization of single-cell subpopulations to better understand disease etiology is paramount. It is thus an opportune time to explore techniques that allow molecular analysis of single cells and to better understand the basis of pathogenesis of diseases like cancer. Single-cell western blotting is one such method that allows analysis of single cells at the protein level. In contrast to traditional western blotting, which relies heavily on bulk analysis of lysates generated from tissues and is often indicative of the population average, this technique allows analysis of lysates from single-cell subpopulations thereby providing a glimpse into cell heterogeneity. The method entails the use of a chip containing 30 µm thick photoactivated polyacrylamide gel spotted with nearly 6400 microwells. Single cells loaded on the chip are captured in the microwells by passive gravity and are then lysed and electrophoresed using the MILO™ single-cell western platform. This method forgoes the use of transfer of proteins on a PVDF and a nitrocellulose membrane, as performed in traditional western blotting, and all other steps including probing of primary and fluorescent secondary antibodies against the protein of interest are performed directly on the chip. The proteins of interest can then be visualized by scanning a chip with the use of a microarray scanner. The entire procedure can be performed in as less as 4-6 h, and thus this method provides several advantages over traditional western blotting.

Toward the Development of Simplified Lateral Flow Assays Using Hydrogels as the Universal Control Line.

Lateral flow assays (LFA) have been widely utilized as point-of-care testing devices in diverse fields. However, it is imperative to preprint costly bioreceptors onto the lateral flow nitrocellulose membrane at the control line. The complex manufacturing process and relatively limited detection capabilities of LFA have impeded their utilization in more challenging fields. Here, we propose a novel and simple strategy to simplify the manufacture of LFA while simultaneously improving the sensitivity by modifying the hydrogel line (HL). In our study, it was observed that the sensitivity of commercial LFA strips could be enhanced by 2-5-fold by incorporating an extra HL. Particularly, a universal control line was developed to accommodate multiple LFA detection modes by substituting the conventional antibody control line with a hydrogel control line (HCL). As a proof of concept, the HCL performance could be associated with the slowdown and interception effect toward fluid, which are dependent on the permeation and hydrophilicity of the hydrogel with varying concentrations in the nitrocellulose membrane. This new design builds the foundation to enhance the sensitivity and develop the simplified LFA sensing platform without additional complicated processes.

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.

A NitroPure Nitrocellulose Membrane-Based Grapevine Virus Sampling Kit: Development and Deployment to Survey Japanese Vineyards and Nurseries.

We developed a NitroPure Nitrocellulose (NPN) membrane-based method for sampling and storing grapevine sap for grapevine virus detection. We devised an efficient nucleic acid extraction method for the NPN membrane, resulting in 100% amplification success for grapevine leafroll-associated virus 2 (GLRaV2) and 3 (GLRaV3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A, grapevine virus B, and grapevine red blotch virus (GRBV). This method also allowed the storage of recoverable nucleic acid for 18 months at room temperature. We created a sampling kit to survey GLRaV2, GLRaV3, and GRBV in Japanese vineyards. We tested the kits in the field in 2018 and then conducted mail-in surveys in 2020-2021. The results showed a substantial prevalence of GLRaV3, with 48.5% of 132 sampled vines being positive. On the other hand, only 3% of samples tested positive for GLRaV2 and none for GRBV.

Case report of self-improving collodion ichthyosis in the newborn.

Self-improving collodion ichthyosis (SICI) is a relatively rare subtype of autosomal recessive congenital ichthyosis (ARCI) that is often characterized by a collodion baby (CB) phenotype at birth. A newborn girl, just 1 hour old, presented with taut, shiny, thick yellow crusts, like parchment, and scales on her trunk and upper limbs. The tightening effect had caused both upper eyelids to appear everted, and her lips and auricles were deformed. Based on whole-exome sequencing and examination of the clinical phenotype, the patient was diagnosed with ARCI. After admission, the exposed mucosa was covered with a sterile Vaseline gauze dressing, and she was placed in an incubator set to a temperature of 32°C with a humidity level of 75%. One week later, the parchment-like scales had begun to flake off, and at the age of 3 weeks, all bodily skin appeared normal. SICI was diagnosed. After discharge, the patient was followed up to 3 months of age, at which time her growth and development were comparable to those of her peers. Clinicians should consider SICI as a possible diagnosis when analyzing the prognosis of patients with CB. Reducing water loss and maintaining the electrolyte balance are particularly important for SICI treatment.

Nitrocellulose membranes in situ grown with Prussian blue nanoparticles as stable nanozyme pads for colorimetric detection of dopamine.

Prussian blue (PB) is a typical peroxidase mimic with simple preparation, low cost and high eco-friendliness, but it still has drawbacks of poor stability (e.g., decomposition in aqueous dispersions) and intrinsic optical interference (e.g., high extinction coefficient over a wide wavelength range) in colorimetric assays. Herein, we used nitrocellulose (NC) membranes as synthesis hosts of PB nanoparticles (NPs) to develop a new type of three-dimensional (3D) porous nanozyme pad. By means of an in situ synthesis route, PB NPs were uniformly grown on the surfaces of the fiber scaffolds with desirable stability, which also avoided signal interference from PB NPs owing to the easy handling of the pads in a quantitative solid state. The pads showed significant peroxidase-mimicking activity toward the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) with the output of colorimetric signals. Based on the reduction of oxidized TMB (oxTMB) by dopamine (DA), the pads were exploited for simple and quantitative colorimetric detection of DA with a limit of detection (LOD) of 0.59 µM and a satisfactory accuracy for analysis of real human urine samples. This is the first attempt at exploiting NC membranes as the synthesis hosts to develop nanozyme pads, which solves the above drawbacks of traditional PB-based peroxidase mimics and has promise for various colorimetric bioanalyses, given the structural benefits of NC membranes and their broad applications in biosensors.

Vertical Flow Immunoassay Based on Carbon Black Nanoparticles for the Detection of IgG against SARS-CoV-2 Spike Protein in Human Serum: Proof-of-Concept.

Point-of-care tests play an important role in serological diagnostics of infectious diseases and post-vaccination immunity monitoring, including in COVID-19. Currently, lateral flow tests dominate in this area and show good analytical performance. However, studies to improve the effectiveness of such tests remain important. In comparison with lateral flow tests, vertical flow immunoassays allow for a reduction in assay duration and the influence of the hook effect. Additionally, the use of carbon black nanoparticles (CNPs) as a color label can provide a lower detection limit (LOD) compared to conventional colloidal gold. Therefore, we have developed a vertical flow immunoassay for the detection of IgG against SARS-CoV-2 spike protein in human serum samples by applying a conjugate of CNPs with anti-human IgG mouse monoclonal antibodies (CNP@MAb). The vertical flow assay device consists of a plastic cassette with a hole on its top containing a nitrocellulose membrane coated with spike protein and an absorbent pad. The serum sample, washing buffer, and CNP@MAb flow vertically through the nitrocellulose membrane and absorbent pads, reducing assay time and simplifying the procedure. In positive samples, the interaction of CNP@MAb with anti-spike antibodies leads to the appearance of black spots, which can be visually detected. The developed method allows for rapid visual detection (5-7 min) of IgG vs. spike protein, with a LOD of 7.81 BAU/mL. It has been shown that an untrained operator can perform the assay and visually evaluate its results. Thus, the presented assay can be used in the further development of test systems for the serological diagnostics of COVID-19 or post-vaccination immunity monitoring.

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.