Identification of new human mesenchymal stem cell biomarker by aptamers / Identificação de novos biomarcadores de células tronco mesenquimais de origem humana por meio de aptâmeros

Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies

Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC

Recent Progress in Aptamer Discoveries and Modifications for Therapeutic Applications.

Aptamers are oligonucleotide sequences with a length of about 25-80 bases which have abilities to bind to specific target molecules that rival those of monoclonal antibodies. They are attracting great attention in diverse clinical translations on account of their various advantages, including prolonged storage life, little batch-to-batch differences, very low immunogenicity, and feasibility of chemical modifications for enhancing stability, prolonging the half-life in serum, and targeted delivery. In this Review, we demonstrate the emerging aptamer discovery technologies in developing advanced techniques for producing aptamers with high performance consistently and efficiently as well as requiring less cost and resources but offering a great chance of success. Further, the diverse modifications of aptamers for therapeutic applications including therapeutic agents, aptamer-drug conjugates, and targeted delivery materials are comprehensively summarized.

Implementation of High-Throughput Sequencing (HTS) in Aptamer Selection Technology.

Aptamers are nucleic acid ligands that bind specifically to a target of interest. Aptamers have gained in popularity due to their high potential for different applications in analysis, diagnostics, and therapeutics. The procedure called systematic evolution of ligands by exponential enrichment (SELEX) is used for aptamer isolation from large nucleic acid combinatorial libraries. The huge number of unique sequences implemented in the in vitro evolution in the SELEX process imposes the necessity of performing extensive sequencing of the selected nucleic acid pools. High-throughput sequencing (HTS) meets this demand of SELEX. Analysis of the data obtained from sequencing of the libraries produced during and after aptamer isolation provides an informative basis for precise aptamer identification and for examining the structure and function of nucleic acid ligands. This review discusses the technical aspects and the potential of the integration of HTS with SELEX.

Label- and modification-free-based in situ selection of bovine serum albumin specific aptamer.

Systematic evolution of ligands by exponential enrichment is a traditional approach to select aptamer, which has a great potential in biosensing field. However, chemical modifications of DNA library or targets before selection might block the real recognition and binding sites between aptamers and their targets. In this study, a label- and modification-free-based in situ selection strategy was developed to overcome this limitation. The strategy is an attempt to screen bovine serum albumin aptamers according to the principle of electrophoretic mobility shift assay, and allowed single-stranded DNA sequence to be fully exposed to interact with bovine serum albumin which was mixed with the agarose gel beforehand. After eight rounds of selection, specific aptamer with low dissociation constant (Kd ) value of 69.44 ± 7.60 nM was selected and used for subsequent establishment of fluorescence biosensor. After optimization, the optimal aptasensor exhibited a high sensitivity toward bovine serum albumin with a limit of detection of 0.24 ng/mL (linear range from 1 to 120 ng/mL). These results indicated that the label- and modification-free-based in situ selection strategy proposed in this work could effectively select specific aptamer to develop aptasensor for sensitive detection of bovine serum albumin or other targets in actual complicated samples.

Nucleic Acid Aptamers as Emerging Tools for Diagnostics and Theranostics.

Aptamers are ssDNA or RNA sequences (20-80 nucleotides) generated in vitro by SELEX (Systematic Evolution of Ligands using EXponential enrichment) against diverse range of targets from small molecules to bacteria, viruses, and even eukaryotic cells. Aptamers, also known as chemical bodies, bind to their respective targets with tunable affinity and specificity, making aptamers as potent probes for diagnostics and excellent ligands for drug delivery in therapeutics. In this chapter, we have described the methods for generating DNA aptamers against proteins and their use in theranostics.

Aptamer Selection for Detecting Molecular Target Using Cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) Technology.

Cell-SELEX is a live cell-based in vitro selection method that generates functional oligonucleotides, or aptamers. Often referenced as the chemist's antibody, aptamers bind to select targets with high affinity and can be utilized in a number of applications, including biomedicine, bioimaging, and biosensing. Here we describe the cell-SELEX technique and discuss this methodology's unique merit(s)-namely the ability to isolate highly selective aptamer panels with no prior knowledge of cellular signatures. This strategy thus presents as a technology that has the potential to enhance the precision of molecular medicine and targeted therapeutics.

Encoded particle microfluidic platform for rapid multiplexed screening and characterization of aptamers against influenza A nucleoprotein.

Aptamers represent interesting bioreceptor alternatives to antibodies when developing a bioassay and are selected by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. After selection, an extensive characterization process is essential to verify the binding affinity and specificity of aptamer candidates, which is the most time-consuming and costly step. In this study, we assessed a new microfluidic platform, namely Evalution™, as a rapid and high throughput aptamer characterization platform. To do this, we first selected aptamers against influenza A nucleoprotein (infA NP) by performing magnetic bead-based SELEX. The selected aptamer candidates were subsequently screened using Evalution™ for their binding kinetics and specificity towards infA NP. All aptamers showed dissociation constants (KD) in the low nanomolar range (from 13 to 41 nM), and differential binding behavior towards control proteins, such as BSA and influenza B nucleoprotein (infB NP). Among 5 selected candidates, one aptamer (NP5) exhibited a significant discrimination between infA NP and infB NP and was further used to benchmark the kinetic analysis of Evalution™ (KD = 41 nM) with an SPR platform (KD = 17 nM). These results suggested that NP5 has the potential to be used for developing sensitive and infA NP specific aptamer-based assay. Moreover, the presented platform proved to be an efficient aptamer characterization tool for performing typical aptamer characterization experiments like binding kinetics (due to the real-time monitoring feature) and specificity assessment in a high-throughput manner due to the multiplexing capacity.

An integrated microfluidic platform to perform uninterrupted SELEX cycles to screen affinity reagents specific to cardiovascular biomarkers.

As cardiovascular diseases (CVD) are responsible for millions of deaths annually, there is a need for rapid and sensitive diagnosis of CVD at earlier stages. Aptamers generated by systematic evolution of ligands by exponential enrichment (SELEX) processes have been shown to be superior to conventional antibody-based cardiac biomarker detection. However, SELEX is a complicated, lengthy procedure requiring multiple rounds of extraction/amplification and well-trained personnel. To circumvent such issue, we designed an automated, miniaturized SELEX platform for the screening of aptamers towards three protein biomarkers associated with CVDs: N-terminal pro-peptide of B-type natriuretic peptide, human cardiac troponin I, and fibrinogen. The developed microfluidic platform was equipped with microfluidic devices capable of sample transport and mixing along with an on-chip nucleic acid amplification module such that the entire screening process (5 rounds of selection in 8 h.) could be performed consecutively on a single chip while consuming only 35 µL of reagents in each cycle. This system may therefore serve as a promising, sensitive, cost-effective platform for the selection of aptamers specific for CVD biomarkers.

Magnetic Separation-Based Multiple SELEX for Effectively Selecting Aptamers against Saxitoxin, Domoic Acid, and Tetrodotoxin.

In this study, a novel magnetic separation-based multiple systematic evolution of ligands by exponential enrichment (SELEX) was applied to select aptamers simultaneously against three kinds of marine biotoxins, including domoic acid (DA), saxitoxin (STX), and tetrodotoxin (TTX). Magnetic reduced graphene oxide (MRGO) was prepared to adsorb unbound ssDNAs and simplify the separation step. In the multiple SELEX, after the initial twelve rounds of selection against mixed targets and the subsequent four respective rounds of selection against each single target, the three resulting ssDNA pools were cloned, sequenced, and analyzed. Several aptamer candidates were selected and subjected to the binding affinity and specificity test. Finally, DA-06 ( Kd = 62.07 ± 19.97 nM), TTX-07 ( Kd = 44.12 ± 15.38 nM), and STX-41 ( Kd = 61.44 ± 23.18 nM) showed high affinity and good specificity for DA, TTX, and STX, respectively. They were also applied to detect and quantify DA, TTX, and STX successfully. The other two multitarget aptamers, DA-01 and TTX-27, were also obtained, which can bind with either DA or TTX. These aptamers provide alternative recognition molecules to antibodies for biosensor applications.

Highly Efficient and Reliable DNA Aptamer Selection Using the Partitioning Capabilities of ddPCR: The Hi-Fi SELEX Method.

In addition to its growing use in detecting and quantifying genes and larger genomic events, the partitioning used in digital PCR can serve as a powerful tool for high-fidelity amplification of synthetic combinatorial libraries of single-stranded DNA. Sequence-diverse libraries of this type are used as a basis for selecting tight-binding aptamers against a specific target. Here we provide a detailed description of the Hi-Fi SELEX protocol for rapid and efficient DNA aptamer selection. As part of that methodology, we describe how Hi-Fi SELEX gains advantages over other aptamer selection methods in part through the use of the massive partitioning capability of digital PCR.

Aptamer-Based Lateral Flow Test Strip for Rapid Detection of Zearalenone in Corn Samples.

An aptamer-based lateral flow test strip was developed for the detection of zearalenone (ZEN). This assay was based on the competition for the aptamer between ZEN and its complementary sequence. Several experimental conditions that could influence sensitivity have been investigated, including the concentration of aptamer and NaCl used in the probe preparation, the mole ratio of streptavidin and biotinylated DNA used in the preparation of test line and control line, and the loading quantity of gold nanoparticles-aptamer conjugates (AuNPs-Apt). Under the optimal experimental conditions, we successfully detected ZEN within a detection range of 5-200 ng/mL and the visual limit of detection of 20 ng/mL. This aptamer-based strip was successfully applied to the determination of ZEN in spiked corn samples, and the recoveries were from 93.4% to 114.2%. All detections can be achieved within 5 min. The results demonstrated that the developed aptamer-based lateral flow test strip is a potential alternative tool for the rapid and sensitive detection of ZEN.

An Enzyme-Linked Aptamer Sorbent Assay to Evaluate Aptamer Binding.

Nucleic acid aptamers are a class of alternative ligands increasingly growing in importance in the face of contemporary detection challenges. Aptamers offer multiple advantages over traditional ligands like antibodies; however, their ability to specifically bind target molecules must first be confirmed after their generation. Use of a plate-based enzyme-linked aptamer sorbent assay (ELASA) is a generally rapid way to screen and characterize aptamer binding to protein targets. ELASA involves directly plating a protein target onto a nonspecific (polystyrene) surface and assessing binding of functionalized (biotinylated) aptamers to those plated proteins using an enzyme conjugate that recognizes the aptamers. Here, we describe an ELASA that was designed and used to evaluate and compare binding of ssDNA aptamers against the capsids of different strains of human norovirus.

Selection and Application of ssDNA Aptamers against Clenbuterol Hydrochloride Based on ssDNA Library Immobilized SELEX.

Clenbuterol hydrochloride (CLB) is often abused as additive feed for livestock to decrease adipose tissue deposition and to increase growth rate. It raises a potential risk to human health through the consumption of animal product. In this study, aptamers with higher affinity and specificity were screened through 16 selection rounds based on the ssDNA library immobilized systematic evolution of ligands by exponential enrichment (SELEX) technique. After cloning and sequencing, five aptamer candidates were picked out for affinity and specificity assays based on a graphene oxide (GO) adsorption method. The results showed that the aptamer CLB-2 binds specifically against CLB with a dissociation constant, Kd, value of 76.61 ± 12.70 nM. In addition, an aptamer-based fluorescence bioassay was established for CLB analysis. The correlation between the CLB concentration and fluorescent signal was found to be linear within the range of 0.10 to 50 ng/mL with a limit of detection of 0.07 ng/mL. It has been further applied for the determination of CLB in pork samples, showing its great potential for sensitive analysis in food safety control.

An Integrated Microfluidic SELEX Approach Using Combined Electrokinetic and Hydrodynamic Manipulation.

This article presents a microfluidic approach for the integration of the process of aptamer selection via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs bead-based biochemical reactions in which affinity-selected target-binding oligonucleotides are electrokinetically transferred for amplification, while the amplification product is transferred back for affinity selection via pressure-driven fluid flow. The hybrid approach simplifies the device design and operation procedures by reduced pressure-driven flow control requirements and avoids the potentially deleterious exposure of targets to electric fields prior to and during affinity selection. In addition, bead-based reactions are used to achieve the on-chip coupling of affinity selection and amplification of target-binding oligonucleotides, thereby realizing on-chip loop closure and integration of the entire SELEX process without requiring offline procedures. The microfluidic approach is thus capable of closed-loop, multiround aptamer enrichment as demonstrated by selection of DNA aptamers against the protein immunoglobulin E with high affinity ( KD = 12 nM) in a rapid manner (4 rounds in approximately 10 h).

Highly Multiplexed RNA Aptamer Selection using a Microplate-based Microcolumn Device.

We describe a multiplexed RNA aptamer selection to 19 different targets simultaneously using a microcolumn-based device, MEDUSA (Microplate-based Enrichment Device Used for the Selection of Aptamers), as well as a modified selection process, that significantly reduce the time and reagents needed for selections. We exploited MEDUSA's reconfigurable design between parallel and serially-connected microcolumns to enable the use of just 2 aliquots of starting library, and its 96-well microplate compatibility to enable the continued use of high-throughput techniques in downstream processes. Our modified selection protocol allowed us to perform the equivalent of a 10-cycle selection in the time it takes for 4 traditional selection cycles. Several aptamers were discovered with nanomolar dissociation constants. Furthermore, aptamers were identified that not only bound with high affinity, but also acted as inhibitors to significantly reduce the activity of their target protein, mouse decapping exoribonuclease (DXO). The aptamers resisted DXO's exoribonuclease activity, and in studies monitoring DXO's degradation of a 30-nucleotide substrate, less than 1 µM of aptamer demonstrated significant inhibition of DXO activity. This aptamer selection method using MEDUSA helps to overcome some of the major challenges with traditional aptamer selections, and provides a platform for high-throughput selections that lends itself to process automation.

A sandwich dipstick assay for ATP detection based on split aptamer fragments.

Aptamer-based strip assay is an easy, highly efficient and low-cost detection method, which has been developed and easily applied to onsite detection. A new sensitive sandwich dipstick assay for adenosine triphosphate (ATP) detection was successfully developed based on specific recognition between split aptamer fragments and the target. In this method, the thiolated aptamer was first conjugated to the surface of gold nanoparticles (AuNPs), while the biotin-aptamer was immobilized on the surface of a nitrocellulose filter in the test line. In the presence of ATP, the thiol-aptamer/ATP/biotin-aptamer complexes were generated, which led to an obvious increase in optical signals at the test line. Under the optimal determination conditions, an excellent linear logarithmic response to the ATP concentration was obtained within the range of 0.5 µM to 5 mM. The limit of detection (LOD) of 0.5 µM was reached at a signal-to-noise ratio of 3. The dipstick assay showed a good average recovery of 96-108 % with the RSD of less than 20 % in urine samples. The proposed method exhibited high specificity against other nucleotides such as the uridine triphosphate (UTP), cytidine triphosphate (CTP), and guanosine triphosphate (GTP). The results indicated that the dipstick strip may be considered as an inexpensive screening tool for onsite ATP determination. Graphical Abstract A simple split aptamer fragments based sandwich-type dipstick assay was developed for ATP detection.

Replacing antibodies with aptamers in lateral flow immunoassay.

Aptamers have been identified against various targets as a type of chemical or nucleic acid ligand by systematic evolution of ligands by exponential enrichment (SELEX) with high sensitivity and specificity. Aptamers show remarkable advantages over antibodies due to the nucleic acid nature and target-induced structure-switching properties and are widely used to design various fluorescent, electrochemical, or colorimetric biosensors. However, the practical applications of aptamer-based sensing and diagnostics are still lagging behind those of antibody-based tests. Lateral flow immunoassay (LFIA) represents a well established and appropriate technology among rapid assays because of its low cost and user-friendliness. The antibody-based platform is utilized to detect numerous targets, but it is always hampered by the antibody preparation time, antibody stability, and effect of modification on the antibody. Seeking alternatives to antibodies is an area of active research and is of tremendous importance. Aptamers are receiving increasing attention in lateral flow applications because of a number of important potential performance advantages. We speculate that aptamer-based LFIA may be one of the first platforms for commercial use of aptamer-based diagnosis. This review first gives an introduction to aptamer including the selection process SELEX with its focus on aptamer advantages over antibodies, and then depicts LFIA with its focus on aptamer opportunities in LFIA over antibodies. Furthermore, we summarize the recent advances in the development of aptamer-based lateral flow biosensing assays with the aim to provide a general guide for the design of aptamer-based lateral flow biosensing assays.

Food sensing: selection and characterization of DNA aptamers to Alicyclobacillus spores for trapping and detection from orange juice.

The quality of the beverage industry's products has to be constantly monitored to fulfill consumers' high expectations. The thermo-acidophilic Gram-positive Alicyclobacillus spp. are not pathogenic, but their heat-resistant endospores can survive juice-processing conditions and have become a major economic concern for the fruit juice industry. Current detection methods rely on cultivation, isolation, and organism identification, which can take up to a week, resulting in economic loss. This work presents the selection and identification of DNA aptamers targeting Alicyclobacillus spores by spore-SELEX (systematic evolution of ligands by exponential enrichment) in orange-juice-simulating buffer. The selection process was verified by various techniques, including flow cytometric binding assays, radioactive binding assays, and agarose gel electrophoresis. The subsequent aptamer characterization included the determination of dissociations constants and selectivity by different techniques, such as surface plasmon resonance spectroscopy and fluorescence microscopy. In summary, 10 different aptamers with an affinity to Alicyclobacillus spp. have been developed, analyzed, and characterized in terms of affinity and specificity.

DNA aptamers against the Cry j 2 allergen of Japanese cedar pollen for biosensing applications.

Sensing pollen allergens is required to prevent allergic disorders such as pollinosis. Aptamers, which bind to specific molecules, offer great potential as useful tools for detecting pollen allergens as measures against allergic disorders. Here, we report the identification of DNA aptamers binding to Cry j 2, one of the major allergens in Japanese cedar pollen, and the histochemical sensing of Cry j 2 in ruptured Japanese cedar pollen. DNA aptamers were selected by systematic evolution of ligands by exponential enrichment (SELEX) using nitrocellulose membranes. Through four rounds of SELEX, we identified aptamers binding to Cry j 2. The aptamers generated staining in ruptured Japanese cedar pollen on glass slides without extraction, similar to anti-Cry j 2 antibodies. The staining was compatible with starch localization, in which Cry j 2 is present. An aptamer, CJ2-06, which had high and specific binding ability to Cry j 2 (K(d)=24 nM), detected an amount of Cry j 2 equivalent to that in several tens of micrograms of pollen. Cry j 2 contained in house dust was detected in a spike test. The aptamers identified in this study can be powerful tools for allergen recognition in the practical biosensing of Cry j 2, leading to preventive measures against allergic disorders caused by Japanese cedar pollen.

Selection of aptamers specific for glycated hemoglobin and total hemoglobin using on-chip SELEX.

Blood glycated hemoglobin (HbA1c) levels reflecting average glucose concentrations over the past three months are fundamental for the diagnosis, monitoring, and risk assessment of diabetes. It has been hypothesized that aptamers, which are single-stranded DNAs or RNAs that demonstrate high affinity to a large variety of molecules ranging from small drugs, metabolites, or proteins, could be used for the measurement of HbA1c. Aptamers are selected through an in vitro process called systematic evolution of ligands by exponential enrichment (SELEX), and they can be chemically synthesized with high reproducibility at relatively low costs. This study therefore aimed to select HbA1c- and hemoglobin (Hb)-specific single-stranded DNA aptamers using an on-chip SELEX protocol. A microfluidic SELEX chip was developed to continuously and automatically carry out multiple rounds of SELEX to screen specific aptamers for HbA1c and Hb. HbA1c and Hb were first coated onto magnetic beads. Following several rounds of selection and enrichment with a randomized 40-mer DNA library, specific oligonucleotides were selected. The binding specificity and affinity were assessed by competitive and binding assays. Using the developed microfluidic system, the incubation and partitioning times were greatly decreased, and the entire process was shortened dramatically. Both HbA1c- and Hb-specific aptamers selected by the microfluidic system showed high specificity and affinity (dissociation constant, Kd = 7.6 ± 3.0 nM and 7.3 ± 2.2 nM for HbA1c and Hb, respectively). With further refinements in the assay, these aptamers may replace the conventional antibodies for in vitro diagnostics applications in the near future.