Complementary Aptamer Research Articles

Early detection for carbohydrate antigen-19-9 based on surface enhanced Raman spectroscopy aptamer sensor

BackgroundIncipient screening for cervical cancer is crucial for treatment and improving prognosis. In this work, SERS based aptamer sensor (aptasensor) has been developed and applied to high-sensitivity detection of cervical cancer tumor marker (Carbohydrate antigen19-9 (CA19-9)) with a microporous chip, which was used as surface enhanced Raman scattering (SERS) analysis platform. MethodsThe aptamer of CA19-9 was immobilized on the gold nanodumbbell as a capture probe and the gold nanobipyramid containing complementary aptamer chains of the signal molecule Cy5 was modified as a signal probe. SERS aptasensor was constructed by combining signal probes and capture probes. It caused signal molecule Cy5 were sandwiched between the signal probes and capture probes; the constructed SERS aptasensor generated a strong SERS signal of Cy5. Based on the strong specificity of the aptamer, the CA19-9 aptamer binds specifically to CA19-9 when CA19-9 exist in the detection environment. The signal probe was released, resulting in a decrease in the SERS signal of the detection environment. ResultsUnder the optimal detection conditions, the detection limit of the aptasensor was calculated to be 1.16 × 10−3 U/mL and limit of quantification is equal to 3.87 × 10−3 U/mL. In addition, a standard cervical cancer bearing mouse model was constructed and the SERS spectra of the serum of mice at different stages were measured. The CA19-9 content in the serum of mice at different stages was calculated. Compared with the results of enzyme-linked immunosorbent assay (ELISA), relative error (RE) values (2.54 %, −6.093 %, −4.922 %, and 3.04 %) of the CA19-9 concentrations detected by the two methods on the day of 0, 7, 14 and 28. ConclusionThe proposed SERS aptasensor provides a new and reliable platform for early cervical cancer screening, and its low RE value proves that it will be a promising CA19-9 detection method.

Dye-sensitized NiO photocathode sensor based on signal-sensitive change strategy for MC-LR detection.

A novel photoelectrochemical (PEC) sensor for the detection of microcystic toxins (MC-LR) was developed on the basis of signal-sensitive change strategy. NiO nanoarray as a basic photoactive material was grown directly on the ITO glass electrode via calcination after hydrothermal reaction, while dye N719 was used to sensitize the electrode for enhancing visible light absorption, and the first signal-on stage was obtained. In the meantime, p-type Cu2O was applied as the signal probe attached to probe DNA (DNA2) to improve the sensitivity, and the second "signal-on" stage appeared because of its synergistic effect with NiO nanoarrays. The PEC signal decreases after the target analyte MC-LR is modified on the electrode due to the stronger affinity between MC-LR and its complementary aptamer DNA; part of the Cu2O-DNA2 will dissociate from the electrode. This sensitive signal change strategy allows the detection limit of the MC-LR sensor to be as low as 1.7pM, which offers an optional method for the sensitive and selective detection of other target molecules, with potential applications in environmental monitoring and toxin determination.

Application of MOF Nanoenzyme in Rapid Detection of Food Allergens System

Food allergies are a common immune response that can lead to serious health problems. Rapid and accurate detection of allergens in food is crucial for protecting people’s health and quality of life. In this study, β-lactoglobulin in milk serves as a model allergen, and nucleic acid aptamer-loaded MN patches are utilized as rapid capture elements. Moreover, metal organic frameworks loaded with complementary aptamer sequences are employed as signal amplification elements. The combination of these two elements establishes a rapid visualization detection system for allergens, employing specific microneedle array patches-MOF nanoenzymes. The results show that the combination of TMB+H2O2 +MOF-RC has the highest peroxidase like activity of 1.732. Compared to TMB+H2O2 +MOF, it has increased by 0.547. Meanwhile, the MN-MOF detection method has excellent specificity for the detection of β-Lg. This specific microneedle array patches and MOF nanoenzymes-based allergen rapid detection system demonstrates practical application benefits, providing a viable solution for swiftly detecting food allergens and safeguarding people’s health and quality of life.

SERS detection platform based on a nucleic acid aptamer-functionalized Au nano-dodecahedron array for efficient simultaneous testing of colorectal cancer-associated microRNAs

A surface-enhanced Raman scattering (SERS) detection platform was constructed based on Au nano-dodecahedrons (AuNDs) functionalized with nucleic acid aptamer-specific binding and self-assembly techniques. SERS labels were prepared by modifying Raman signaling molecules and complementary aptamer chains and were bound on the aptamer-functionalized AuNDs array. Using this protocol, the limits of detection (LODs) of miR-21 and miR-18a in the serum were 6.8 pM and 7.6 pM, respectively, and the detection time was 5 min. Additionally, miR-21 and miR-18a were detected in the serum of a mouse model of colorectal cancer. The results of this protocol were consistent with quantitative real-time polymerase chain reaction (qRT-PCR). This method provides an efficient and rapid method for the simultaneous testing of miRNAs, which has great potential clinical value for the early detection of colorectal cancer (CRC).

Concentric interdigitated dielectrodes on silica substrate to monitor neuroprotective effect of anesthetics

General anesthesia is commonly used in major surgeries, in most cases it is completely reversible and central nerve system returns normal after the elimination of anesthetic agent. At the same time, usage of anesthetic agents may result in a long-lasting neurological effect due to increasing level of neural injury biomarker. Various neuro-proteins have been identified to provide a clear insight of neurological injury, among them, tau protein level fluctuates during the pre- and post-operative periods, which leads to different neurological complications, such as Parkinson’s and Alzheimer’s diseases. This research was focused to develop a sensitive tau protein biosensor on modified concentric interdigitated dielectrode surface and measured by impedance spectroscopy. The sensing surface with silica substrate and embedded gold dielectrodes were used for aptamer-antibody sandwich assay and attained the detection limit of tau protein to 1 fM on a linear regression range until 1 nM (R2 = 0.9899; y = 535.04x −398). Further, selective experiments in tau protein spiked artificial cerebrospinal fluid increases the current responses with the concomitant increment of tau protein. Experiments with complementary aptamer, amyloid-β40 and recombinant human complement failed to response. This tau biosensor helps to quantify tau protein levels silica-gold hybrid surface and monitor the neuroprotective effect of anesthetics.

Gold structured nanourchin as anesthetic biosensor for determining neuroprotective effect

Research has proven that confusion, delirium, and cognitive dysfunction are the common symptoms caused by anesthesia. In general, neuroproteins fluctuation is common during the process of anesthetic treatment. Among others, tau protein was found to the higher fluctuations after the anesthetic process, and it is highly associated with various neuro-related diseases. Monitoring the level of tau protein helps to identify the neuro-related problems during and after the anesthetic processes. This research was focused on developing a tau electrochemical impedance biosensor on aptamer and antibody-modified electrodes. Aptamer and antibody were immobilized on the gold nanourchin with a chemical linker. Gold nanourchin enhances the probe of aptamer and antibody attachment on the electrode and improves the tau protein detection as low as 1 fM [y = 1.5604x − 1.5643; R2 = 0.9681], determined on a linear range from 1 fM to 1 nM. Further, control performances with complementary aptamer and non-immune antibodies fail to change the response of the current, indicating the specific detection of tau protein. This tau sensor detects the tau protein in its lower level and helps to identify the neuro-related problem caused by anesthetics.

Analysis of human epidermal growth factor receptor 2 interaction on aptamer-probed interdigitated electrode for breast cancer diagnosis.

Breast cancer has been reported to be high in its incidence with women, and early identification of breast cancer helps to improve and provide an effective treatment. Tumor markers are active substances; in particular, human epidermal growth factor receptor 2 (HER2) is over-expressed at the level of 20%-30%. This research work developed a highly sensitive HER2 biosensor on the interdigitated electrode (IDE) by using aptamer as a detection probe. To enhance the analytical performances, aptamer was attached to the gold nanoparticle and immobilized on the IDE through a chemical linker [(3-aminopropyl)triethoxysilane]. On the aptamer conjugation, HER2 was quantified through current-volt measurements, and the limit of detection of HER2 was calculated as 1pg/mL on a linear range from 0.1 to 3000pg/mL at an R2 (regression coefficient) of 0.9657. Further, a selective performance with human serum increased the current responses by increasing HER2 concentrations. Specific experiments with control protein and complementary aptamer sequence failed to enhance the current responses. This HER2 biosensor reflects the occurrence of breast cancer at its lower abundance and helps to identify the associated complications.

Uncovering Adenosine‐Specific Signals: A Label‐Free Aptamer‐Based Approach with Gold Nanoparticles for Specific Detection

AbstractWe present a novel colorimetric aptasensing method for specific adenosine (Ade) detection based on complementary aptamer hybridization and gold nanoparticles (AuNPs). Unlike traditional approaches, our method overcomes limitations of selectivity and specificity by incorporating an additional step involving the complementary strand of the aptamer (c‐Ade aptamer) after incubating Ade with the aptamer. In the absence of Ade, the c‐Ade aptamer hybridizes with the aptamer, causing AuNPs aggregation and a blue color change. However, in the presence of Ade, the aptamer‐bound target prevents hybridization, allowing the free c‐Ade aptamer to adsorb onto the AuNPs, resulting in a red color change. This unique color reversal distinguishes our approach from conventional label‐free methods. We optimized the detection conditions, including incubation time and salt concentration, to enhance sensitivity and reproducibility. The calibration curve exhibited a linear relationship between the colorimetric response and Ade concentration, with a limit of detection of 3.0±0.8 nM. Our method demonstrated excellent selectivity for Ade, even in the presence of structurally similar interfering compounds. Furthermore, we successfully applied our approach to detect Ade in complex biological samples with minimal sample preparation. Our colorimetric aptasensing method offers a simple, cost‐effective, and specific tool for detecting targets in various applications.

A ratiometric SERS aptasensor array for human DNA glycosylaseat single-cell sensitivity/resolution

Human 8-oxoguanine DNA glycosylase (hOGG1) is involved in the cellular genomic 8-oxoguanine (8-oxoG) excision repair to maintain genome stability. Accurate detection of hOGG1 activity is essential for clinical diagnosis and treatment of various human pathology. Yet, the quantitative detection of hOGG1 remains challenging for existing methods due to poor reproducibility and portability. Herein, we propose a ratiometric array-based SERS point-of-care testing method for hOGG1 activity. A kind of reproducible, uniform and stable plasmonic multi-microarray reaction cells was constructed by assembling AuNPs on the substrate modified by aminosilane and segmented by silica gel gasket, which greatly improved the sensitivity, portability and repeatability of SERS measurement. Based on this, the ratiometric method is further used to effectively overcome the instability of single SERS signal intensity, which allows signal rationing and provides built-in correction for environment effects. In specific, we designed two different Raman-labeled probes for the detection of hOGG1, a thiol- and Cy3-labeled aptamer as an internal standard and a Rox-labeled 8-oxoG-modified complementary aptamer as a signal probe. The ratio value between Cy3 and Rox SERS intensity is well linear with the hOGG1 activity on logarithmic scales in the range from 5 × 10−5 to 5 × 10−3 U/mL, and the limit of detection reaches 3.3 × 10−5 U/mL. Moreover, this strategy can be applied for the screening of inhibitors and the monitoring of cellular hOGG1 activity fluctuation at single-cell levels, providing a flexible and adaptive tool for clinical diagnosis, biochemical processes and drug discovery.

Sandwich biosensing on a nanodiamond-modified interdigitated electrode for monitoring the occurrence of osteosarcoma

BackgroundOsteosarcoma is a highly heterogeneous manifestation with varied subtypes and is diagnosed by imaging techniques; however, quantification of blood-based biomarkers boosts genuine osteosarcoma identification. MethodsHerein, a sensitive sandwich pattern with aptamer and antibody was conducted on a nanodiamond-modified interdigitated electrode surface to identify osteopintin, a well-suited biomarker for osteosarcoma. Diamond nanoparticles measured as 5±3 nm by a high-resolution microcopy were attached to the sensing surface through chemical [(3-aminopropyl)triethoxysilane, 1,1′-carbonyldiimidazole] and biological (biotin-streptavidin) linkers to immobilize the aptamer on the nanodiamond. The detection limit of osteopontin was attained to be 100 pM in phosphate-buffered saline (PBS)-spiked samples using a sandwich, and it was 1 nM with only aptamer at the coefficient determinations R2 values of 0.99 (y=1.2106x-1.0127) and 0.98 (y=1.0086x-0.7867), respectively, on linear concentrations of 0.1-100 nM osteopontin. Furthermore, biofouling experiments with complementary aptamer sequences, albumin and control samples did not increase the current responses, confirming the specific identification of osteopontin, which was further evidenced in serum-containing samples. Significant FindingsThis biosensing assay quantifies osteopontin at lower levels and diagnoses osteosarcoma.

An Alkyne-Mediated SERS Aptasensor for Anti-Interference Ochratoxin A Detection in Real Samples.

Avoiding interference and realizing the precise detection of mycotoxins in complex food samples is still an urgent problem for surface-enhanced Raman spectroscopy (SERS) analysis technology. Herein, a highly sensitive and specific aptasensor was developed for the anti-interference detection of Ochratoxin A (OTA). In this aptasensor, 4-[(Trimethylsilyl) ethynyl] aniline was employed as an anti-interference Raman reporter to prove a sharp Raman peak (1998 cm-1) in silent region, which could avoid the interference of food bio-molecules in 600-1800 cm-1. 4-TEAE and OTA-aptamer were assembled on Au NPs to serve as anti-interference SERS probes. Meanwhile, Fe3O4 NPs, linked with complementary aptamer (cApts), were applied as capture probes. The specific binding of OTA to aptamer hindered the complementary binding of aptamer and cApt, which inhibited the binding of SERS probes and capture probes. Hence, the Raman responses at 1998 cm-1 were negatively correlated with the OTA level. Under the optimum condition, the aptasensor presented a linear response for OTA detection in the range of 0.1-40 nM, with low detection limits of 30 pM. In addition, the aptasensor was successfully applied to quantify OTA in soybean, grape and milk samples. Accordingly, this anti-interference aptasensor could perform specific, sensitive and precise detection of OTA in real samples, and proved a reliable reference strategy for other small-molecules detection in food samples.

A Zeolite Nanoparticle-Modified Anionic Surface for Aptasensing Lipocalin-2 in Ulcerative Colitis by Dual-Electrodes.

An aptasensor was developed on an interdigitated microelectrode (IDME) by current-volt sensing for the diagnosis of ulcerative colitis by detecting the biomarker lipocalin-2. Higher immobilization of the anti-lipocalin-2 aptamer as a probe was achieved by using sodium dodecyl benzenesulfonate-aided zeolite particles. FESEM and FETEM observations revealed that the size of the zeolite particles was <200 nm, and they displayed a uniform distribution and spherical shape. XPS analysis attested the occurrence of Si, Al, and O groups on the zeolite particles. Zeolite particles were immobilized on IDME by a (3-aminopropyl)-trimethoxysilane amine linker, and then, the aptamer as the probe was tethered on the zeolite particles through a biotin-streptavidin strategy assisted by a bifunctional aldehyde linker. Due to the high occupancy of the aptamer and the efficient electric transfer from zeolite particles, higher changes in current can be observed upon interaction of the aptamer with lipocalin-2. The lower detection of lipocalin-2 was noted as 10 pg/mL, with a linear range from 10 pg/mL to 1 μg/mL and a linear regression equation of y=8E-07x+8E-08; R² = 0.991. Control experiments with complementary aptamer and matrix metalloproteinase-9 indicate the specific detection of lipocalin-2. Furthermore, spiking lipocalin-2 in human serum does not interfere with the identification.

Introducing an SPRi-based titration assay using aptamers for the detection of Legionella pneumophila

Legionella are waterborne bacteria and the causative agents behind a severe respiratory disease known as Legionnaires Disease (LD). Standard ISO-certified methods to detect Legionella (plate count and qPCR) are time-consuming and tedious necessitating the development of novel detection strategies. This study introduces an SPRi-based titration assay that uses L. pneumophila (Lp) aptamers. Multiple parameters including buffer conditions, aptamer concentrations, complementary aptamer probe (cApt) concentrations and incubation times were investigated and evaluated for optimal signal response. For maximum hybridization efficiency on the sensor surface, high salt buffer (SSC) as well 500 nM of immobilized cApt resulted in a maximum signal response. Additionally, it was observed that of the three buffers (PBS, Fraquil, SSC) tested in this assay only SSC facilitated incremental binding of aptamers to Lp such that varying concentrations of Lp could be determined. We were able to detect varying concentrations of Lp down to a limit of 104 cells/ml without any sample processing or signal amplification steps, highlighting the potential of this system for SPRi-based Lp detection. The assay is also specific to Lp and shows negligible detection of Pseudomonas, a common inhabitant of water systems.

Nanomaterial-assisted determination of osteosarcoma by antibody-osteopontin-aptamer sandwich ELISA.

Immobilization and detection of small molecules is one of the challenging tasks in any given sensing system as the dissociation equilibrium constant is higher. Generating a right immobilization system with small molecules is mandatory for developing the drug-discovery process and disease identification. Immobilizing smaller probes on the ELISA plate is challenging because of its less adsorption on the polystyrene (PS) substrate. This research work developed an iron nanomaterial-based linker to attach osteopontin-specific aptamer on PS substrate. Iron oxide nanoparticle was attached on PS plate through amine modification and then antibody was attached by COOH reaction. On the osteopontin-modified plate, osteosarcoma biomarker of osteopontin was identified by its specific antibody and aptamer sandwich with the detection limit of 1nM. Further, biofouling experiments with other molecules, such as lysozyme, and complementary aptamer failed to show the ELISA adsorption signal, indicating the iron oxide nanoparticle-modified PS plate specifically recognizes osteopontin. This research work effectively identifies the lesser abundance of osteopontin and helps to diagnose the osteosarcoma-related problems.

Aptasensing luteinizing hormone to determine gynecological endocrine complications on graphene oxide layered sensor.

Luteinizing hormone (LH)/lutropin is an interstitial cell-stimulating hormone playing a predominant role in the reproductive system, and highly correlated with the infertility treatment in both men and women. This research was concentrated to quantify LH level by using interdigitated electrode sensor. To improve the electric current flow, sensing electrode was modified with graphene oxide (GO) and the aptamer probe was attached on GO through biotin-streptavidin linker. Current responses were measured with aptamer-LH interaction at the target concentrations between 7.5nM and 1μM and the detection limit of LH was calculated as 60nM with the determination coefficient (R2 ) value, 0.9229 [y = 1.296x - 2.8435] on a linear range from 30nM to 1μM. Further, biofouling effect on sensing electrode surface was analyzed with complementary aptamer sequence, control proteins (albumin and globulin). The above GO-aptamer-modified interdigitated electrode sensor helps to quantify LH level and diagnose gynecological endocrinology-related complications.

An iron oxide nanoworm hybrid on an interdigitated microelectrode silica surface to detect abdominal aortic aneurysms.

An abdominal aortic aneurysm (AAA) is abnormal swelling in the abdominal aorta and a prevalent life-threatening disease. This research introduces a new interdigitated microelectrode (IDME)-sensing surface modified by iron oxide nanoworms (IONWs) for detecting the AAA biomarker insulin-like growth factor-1 (IGF1). A sandwich pattern was formulated with the IGF1 aptamer and IGFBP1 (IGF binding protein-1) on the IONW-constructed IDME hybrid to identify IGF1. The surface morphology of the IONWs revealed a uniform distribution of worm-like structures (80-100nm) as confirmed by FESEM and FETEM analyses. Further, the presence of the major elements, Fe and O, was confirmed by EDX and XPS studies. The crystal planes that appeared in the IONW reflect cubic magnetite. IONW-modified IDME attained a limit of detection for IGF1 of 1 fM (3σ) with an aptamer-IGF1-IGFBP1 sandwich. This sandwich with IGFBP1 enhanced the current level at all concentrations of IGF1 and displayed linearity in the range 1 fM to 100 pM with a determination coefficient of R2 = 0.9373 [y = 3.38221x - 4.79]. Control experiments with complementary aptamer sequences, IGF2 and IGFBP3 did not show notable signal changes, indicating the specific detection of IGF1. This IONW constructed electrode helps to achieve the detection of low amounts of IGF1 and diagnose AAA at the stage prior to rupture.

Enhancing erythrocyte-influenza virus specificity by glycan-conjugated gold nanoparticle: Validation of hemagglutination by aptamer and neuraminidases.

This study demonstrated the terminated sialo-sugar chains (Neu5Acα2,6Gal and Neu5Acα2,3Gal)-mediated specificity enhancement of influenza virus and chicken red blood cell (RBC) by hemagglutination assay. These glycan chains were immobilized on the gold nanoparticle (GNP) to withhold the higher numbers. With the preliminary optimization, a clear button formation with 0.5% RBC was visualized. On the other hand, intact B/Tokio/53/99 with 750nM hemagglutinin (HA) displayed a nice hemagglutination. The interference on the specificity of RBC and influenza virus was observed by anti-influenza aptamer at the concentration 31nM; however, there is no hemagglutination prevention was noticed in the presence of complementary aptamer sequences. Spiking GNP-conjugated Neu5Acα2,6Gal or Neu5Acα2,3Gal or a mixture of these two to the reaction promoted the hemagglutination to 63-folds higher with 12nM virus, whereas under the same condition the heat-inactivated viruses were lost the hemagglutination. Neuraminidases from Clostridium perfringens and Arthrobacter ureafaciens at 0.0025 neuraminidase units are able to abolish the hemagglutination. Other enzymes, Glycopeptidase F (Elizabethkingia meningoseptica) and Endoglycosidase H (Streptomyces plicatus) did not show the changes with agglutination. Obviously, sialyl-Gal-terminated glycan-conjugated GNP amendment has enhanced the specificity of erythrocyte-influenza virus and able to be controlled by aptamer or neuraminidases.

An electrochemical sensor for high sensitive determination of lysozyme based on the aptamer competition approach

Abstract Protein is a kind of basic substance that constitutes a life body. The determination of protein is very important for the research of biology, medicine, and other fields. Lysozyme is relatively small and simple in structure among all kinds of proteins, so it is often used as a standard target detector in the study of aptamer sensor for protein detection. In this paper, a lysozyme electrochemical sensor based on aptamer competition mechanism is proposed. We have successfully prepared a signal weakening electrochemical sensor based on the lysozyme aptamer competition mechanism. The carboxylated multi-walled carbon nanotubes (MWCNTs) were modified on the glassy carbon electrode, and the complementary aptamer DNA with amino group was connected to MWCNTs. Because of the complementary DNA of daunomycin into the electrode, the electrochemical signal is generated. When there is a target, the aptamer binds to lysozyme with higher binding power, and the original complementary chain breaks down, resulting in the loss of daunomycin inserted into the double chain and the weakening of electrochemical signal. Differential pulse voltammetry was used to determine lysozyme, the response range was 1–500 nM, the correlation coefficient was 0.9995, and the detection limit was 0.5 nM. In addition, the proposed sensor has good selectivity and anti-interference.

Alzheimer's Disease Determination by a Dual Probe on Gold Nanourchins and Nanohorn Hybrids.

BackgroundAlzheimer’s disease (AD) is a neurodegenerative chronic disorder that causes dementia and problems in thinking, cognitive impairment and behavioral changes. Amyloid-beta (Aβ) is a peptide involved in AD progression, and a high level of Aβ is highly correlated with severe AD. Identifying and quantifying Aβ levels helps in the early treatment of AD and reduces the factors associated with AD.Materials and MethodsThis research introduced a dual probe detection system involving aptamers and antibodies to identify Aβ. Aptamers and antibodies were attached to the gold (Au) urchin and hybrid on the carbon nanohorn-modified surface. The nanohorn was immobilized on the sensor surface by using an amine linker, and then a Au urchin dual probe was immobilized.ResultsThis dual probe-modified surface enhanced the current flow during Aβ detection compared with the surface with antibody as the probe. This dual probe interacted with higher numbers of Aβ peptides and reached the detection limit at 10 fM with R2=0.992. Furthermore, control experiments with nonimmune antibodies, complementary aptamer sequences and control proteins did not display the current responses, indicating the specific detection of Aβ.ConclusionAβ-spiked artificial cerebrospinal fluid showed a similar response to current changes, confirming the selective identification of Aβ.

Aptamer-17β-estradiol-antibody sandwich ELISA for determination of gynecological endocrine function.

17β-Estradiol-E2 (17β-E2) is a steroid hormone that plays a major role in the reproductive endocrine system and is involved in various processes, such as pregnancy, fertility, and menopause. In this study, the performance of an enzyme-linked immunosorbent assay (ELISA) for 17β-E2 quantification was enhanced by using a gold nanoparticle (GNP)-conjugated aptamer. An anti-17β-E2-aptamer-GNP antibody was immobilized on an amine-modified ELISA surface. Then, 17β-E2 was allowed to interact with and be sandwiched by antibodies. Aptamer-GNP conjugation was confirmed by colorimetric assays via the naked eye and UV-visible light spectroscopy. The detection limit based on a signal-to-noise ratio (S/N) of 3 was estimated to be 1.5nM (400pg/mL), and the linear range was 1.5-50nM. Control experiments (without 17β-E2/with a complementary aptamer sequence/with a nonimmune antibody) confirmed the specific detection of 17β-E2. Moreover, 17β-E2 spiking of human serum did not interrupt the interaction between 17β-E2 and its antibody and aptamer. Thus, the developed ELISA can be used as an alternate assay for quantification of 17β-E2 and assessment of endocrine-related gynecological problems.