Gestational hypertension is one of the complicated disorders during pregnancy; it causes the significant risks, such as placental abruption, neonatal deaths, and maternal deaths. Hypertension is also responsible for the metabolic and cardiovascular issues to the mother after the years of pregnancy. Identifying and treating gestational hypertension during pregnancy by a suitable biomarker is mandatory for the healthy mother and foetus development. Cortisol has been found as a steroid hormone that is secreted by the adrenal gland and plays a pivotal role in gestational hypertension. A normal circulating level of cortisol is involved in the regulation of blood pressure, and it is necessary to monitor the changes in the level of cortisol during pregnancy. In this work, aptamer-based colorimetric assay is demonstrated as a model with gold nanorod to quantify the level of cortisol using the coordinated aggregation (at 500 mM of NaCl) and dispersion (with 10
Gestational hypertension or pregnancy-induced hypertension complicates ∼10% of the pregnant cases and causes a poor perinatal outcome. It is also responsible for raising other diseases, such as elevated blood pressure in the artery, preeclampsia, and eclampsia, during the period of pregnancy [
In the current investigation, an aptamer-based colorimetric assay was performed to quantify the level of cortisol. Aptamer, a DNA or RNA molecule, has been generated from the randomized library of molecules by a method “SELEX” (Systematic Evaluation of Ligands by Exponential enrichment) with three vital steps, which includes binding, separation, and amplification [
The visual colorimetric analysis is the salt-induced aggregation assay by utilizing DNA, RNA, or aptamers with the gold nanostructure to detect the desired target. Gold is one of the unavoidable materials in the field of biosensors due to its versatile physical and chemical properties. Moreover, gold nanoparticle (GNP) is smaller, suitable to confine the electrons in order to produce the quantum effects, a key consideration for the colorimetric assay [
Gold nanorod (GNR) was obtained from Nanocs, USA. Sodium chloride (NaCl) was procured from Sigma-Aldrich, USA. The hormones cortisol and progesterone were from Adooq Biosciences (USA). Norepinephrine was from Abcam (USA). Anticortisol sequence was adapted from Sanghavi et al. [
To perform the colorimetric assay, first optimize a suitable concentration of NaCl to induce the aggregation of GNR. Different concentrations of NaCl were added independently with the constant volume of 10
Before performing the colorimetric assay, the condition was optimized for the right aptamer concentration to stabilize the GNR at a high salt concentration. Different concentrations of the diluted aptamer were mixed with 10
The aptamer modified GNR (aptamer-GNR) was used to detect the cortisol. For that, 1 mg/mL of cortisol was added with aptamer-GNR and kept for 30 min at RT. Then, the higher concentration of NaCl was added to the solution to observe the color change. After the confirmation of detection, to evaluate the limit of detection, the cortisol concentrations were titrated from 0.625 to 1 mg/mL by interacting aptamer-GNR. Specific detection of cortisol was carried out with two control hormones, namely norepinephrine and progesterone. For that, 1 mg/mL of control hormone was mixed with aptamer-GNR and incubated for 30 min at RT. Then, NaCl was added to evaluate the interaction of aptamer with control hormones. The results obtained were compared with 1 mg/mL of cortisol interaction with aptamer-GNR.
Gestational hypertension is a critical disorder during the pregnancy period and it causes various issues with foetus development and delivery. Finding a level of hypertension is necessary to take care of the mother and baby during and after the period of pregnancy. Cortisol is the stress hormone and its level plays a crucial role in causing different diseases, such as gestational hypertension, during pregnancy.
In the materials study, it has been widely accepted that the wavelength shift of the plasmon band with the gold shows a big impact in the biosensing applications. In general, the spherical-shaped gold particles have been used for the colorimetric assay to induce a large shift for the high-performance detection, in which the controlled aggregation and dispersion causes the spectral difference and in the presence of the target, aggregation with ionic solution displays a broad spectrum under UV-visible spectroscopy scanning. However, GNP-based colorimetric assay is not suitable for multiplex analysis due to the absence of a properly shaped spectrum. Researchers are looking for an alternate particle to minimize a wide spectrum in order to move towards the multiple target analysis. It has been revealed that the usage of anisotropic silver nanoparticles with tetrahedron shows a spectral shift upon target interaction but it does not cause the aggregation, and demonstrated a microarray for molecular fingerprint analysis. Researchers also proposed the usage of GNR to overcome the high aggregation, as GNR can be fabricated at different range of size ratios and has unique advantage for multiplex analysis. Towards this direction, the current study is an attempt to optimize the condition for future multiplex analysis [
To proceed in this line, the current research has been carried out to detect the level of cortisol by a gold nanorod- (GNR-) based aggregation on colorimetric assay using an aptamer generated against cortisol. Figure
(a) Schematic representation of cortisol detection by aptamer-GNR based colorimetric assay. As-received GNR appears red and in the presence of NaCl, it turned into purple. At higher concentration of aptamer-GNR, it appears to be red even in the presence of NaCl. When aptamer-GNR reacts with cortisol at appropriate concentration, the color of the GNR solution is turned to purple with NaCl, indicating the release of the aptamer from the GNR. The aggregation is displayed by SEM analysis (inset). (b) Secondary structure of anticortisol aptamer. Folded by mfold online software.
Before initiating the detection of cortisol, determination of a suitable concentration of NaCl is necessary to achieve higher sensitivity; for that, different concentrations of NaCl were tested with the constant GNR volume. Figure
NaCl titration on GNR. (a) Different concentrations of (30 to 500 mM) NaCl were mixed independently with a constant amount of GNR and the aggregation pattern was observed. Inset displays the color developments. (b) Peak absorbance maximum with different concentrations of NaCl, averaged with different experimental replicates. Inset is for aggregation obtained by SEM.
Upon finding the optimal concentration at 500 mM of NaCl, the experiment was performed to find a suitable concentration of anticortisol aptamer to cover completely the surface of GNR, to be stable under 500 mM of NaCl in the absence of cortisol. Without the complete coverage, the GNR will cause the aggregation by NaCl even in the absence of cortisol and leads to the erroneous positive result. For the optimization analysis, initially different concentrations from 1.25 to 10
Optimization of aptamer concentration. (a) Aptamer with concentrations of 1.25 to 20
The abovementioned experiments were used to determine the optimal NaCl and aptamer concentrations. Before proceeding further for the cortisol detection, the nonspecific binding of cortisol on the surface of the GNR was tested. If cortisol itself binds on the GNR, it may lead to a false-negative result. For this analysis, different concentrations of cortisol (0.5, 1, 2, and 4 mg/mL) were mixed independently with the constant GNR and induced the color change by 500 mM of NaCl. It was noticed that even when the concentration of cortisol was increased, the color of the GNR solution turned to purple in the presence of NaCl due to the aggregation, which means that the cortisol itself is not able to bind on the surface of the GNR, and similar results were observed with all the concentrations tested (Figure
Nonfouling effect of cortisol on GNR. Different concentrations of cortisol (0.5–4 mg/mL) were mixed independently with constant amount of GNR, and 500 mM NaCl was added to evaluate the nonfouling effect.
After all the optimizations, we performed the colorimetric assay using aptamer, cortisol, and GNR with the above final conditions. Initially, the higher concentration (1 mg/mL) of cortisol was used to evaluate the release of aptamer from the GNR. As shown in Figure
(a) Detection of cortisol by colorimetric assay on aptamer-GNR conjugates. 1 mg/mL of cortisol was mixed with aptamer-GNR and checked the aggregation in the presence of NaCl. Inset displays the graphical representation. (b) Mechanism of dispersion and aggregation mimics the above reaction.
Since it was found that 1 mg/mL of cortisol was clearly detected by the colorimetric assay, to evaluate the limit of detection, the titration was performed with the cortisol from 1 mg/mL down to 0.06 mg/mL under similar experimental conditions. Figure
(a) Limit of detection with cortisol. Cortisol concentrations from 0 to 1 mg/mL were mixed independently with GNR-aptamer conjugates and the aggregation was checked in the presence of NaCl. Inset displays the color developments. (b) Peak absorbance maximums with different concentrations of cortisol, averaged with different experimental replicates. The arrow indicates the direction of the changes.
(a) Linear relationship between the absorption maximum and the concentration of the cortisol. 0 to 1 mg/mL of cortisol were used to detect by using GNR-aptamer conjugates. (b) Specific detection of cortisol was carried out with two different control hormones (norepinephrine (C1) and progesterone (C2)). The aptamers interacted with only cortisol, and the color of the GNR was changed to purple due to the aggregation.
Table
Comparison among the available methods for the quantitative cortisol detection.
Method | Material | Probe | Sensitivity | Reference |
---|---|---|---|---|
Chemiresistor | Graphene | Antibody | 10 pg/mL | [ |
Integrated electrode | MOS2 | Antibody | 1 ng/mL | [ |
Printed electrode sensor | Graphene | Antibody | 0.1 ng/mL | [ |
Electrochemical sensor | Cofired ceramic | Antibody | 10 pg/mL | [ |
Electrochemical impedance spectroscopy | Zinc oxide | Antibody | 1 ng/mL | [ |
Piezoelectric immunosensor | Gold-coated surface | Antibody | 36 |
[ |
RAMAN spectroscopy | — | Antibody | Human serum (ng/mL) | [ |
Electrochemical impedance spectroscopy | Gold electrode | Antibody | 0.5 mg/mL | [ |
Specific detection of cortisol was shown by performing the experiment with two different control hormones namely, norepinephrine and progesterone. As shown in Figure
Gestational hypertension causes various health issues to the mother and baby during and after the period of pregnancy. Identifying the real condition of hypertension with a suitable biomarker is mandatory to treat properly. In this work, cortisol, known as the “stress hormone,” was detected by the colorimetric assay using aptamer and gold nanorod conjugate as the primary tools. Cortisol was clearly detected by showing the color change of the gold nanorod solution turning to purple from red with monovalent salt, and the limit of detection was found as 0.25 mg/mL. This method of detection has advantages over other methods to quantify the levels of cortisol with a higher specificity and helps to treat gestational hypertension.
Gold nanorod
Nanomolar
Nanometer
Millimolar
Optical density
Microliter
11
Systematic Evaluation of Ligands by Exponential Enrichment
Gold nanoparticle
Ribonucleic acid
Deoxyribonucleic acid.
All the data are fully available without restriction.
The authors declare that they have no conflicts of interest.
All the authors contributed to the preparation of the manuscript and discussion. All the authors read and approved the final manuscript.
It includes the detection of cortisol on interdigitated electrode sensor, for the comparative study. It also includes brief method and the obtained results. Figure S1: the interaction of cortisol and aptamer on interdigitated electrode sensor. Reference for the described method is also provided.