Development of an Impedimetric Aptasensor for Detection of Progesterone in Undiluted Biological Fluids

Abstract

A cost-effective, deployable, and quantitative progesterone biosensor is desirable for regular progesterone sensing in biological and environmental samples to safeguard public health. Aptasensors have been shown to be affordable as compared to antibody-based sensors, but so far, none of the progesterone aptamers could detect it in undiluted and unprocessed biological samples. Thus, to select an aptamer suitable for biosensing in unprocessed biological samples, a modified magnetic bead-based approach with counter-selection in milk and serum was performed. G-quadruplex forming progesterone aptamers were preferentially screened through in silico, gold nanoparticle-based adsorption–desorption assay and circular dichroism spectroscopy. GQ5 aptamer showed extended stability and a high progesterone binding affinity (K_D 5.29 ± 2.9 nM) as compared to any other reported progesterone aptamers (P4G11 and P4G13). Under optimized conditions, GQ5 aptamer was coated on the gold electrode to develop an impedimetric aptasensor (limit of detection: 0.53, 0.91, and 1.9 ng/mL in spiked buffer, undiluted milk, and serum, respectively, with the dynamic range of detection from 0.1 to 50 ng/mL in buffer and 0.1 to 30 ng/mL in both milk and serum). The aptasensor exhibited a very high level of κ value (>0.9) with ELISA to detect progesterone in milk and serum. The aptasensor could be regenerated three times and can be stored for up to 10 days at 4 °C. Therefore, GQ5 may be used to develop a portable impedimetric aptasensor for clinical and on-site progesterone sensing in various biological and environmental samples.

Introduction

Progesterone (P₄) is a small steroid biomolecule that has medical, veterinary, and public health significance.^1,2 The cyclical change in its expression during the reproductive cycle plays an essential role in the development and maturation of oocytes.³ An aberrant level of progesterone in women is predictive of unsuccessful pregnancy associated with abnormal uterine bleeding, early embryonic mortality, ectopic pregnancy, and so forth.⁴ In farm animals, high and low levels of progesterone in milk or serum have been used to confirm the pregnancy and the time for breeding.⁵ A lower level of progesterone negatively correlates with conception in both humans and animals.⁶ Furthermore, a high level of progesterone has also been reported in water bodies polluted by industrial effluents. Consumption of such water by human, animal, and aquatic life leads to several reproductive complications, including infertility as it acts as an endocrine disruptor.⁷ To avoid such adverse environmental consequences and for regular monitoring of human and animal reproductive health, the development of a cost-effective, deployable, and quantitative biosensor for progesterone is desirable. Such a biosensor can be used for frequent screening of the environmental and clinical samples for progesterone detection even in resource-limited settings and remote areas.

Aptamers are being used as an affordable alternative to antibody-based sensors as they form thermodynamically favored nanostructures by Watson–Crick base pairing.⁸ Aptasensors have been developed for the detection of progesterone but have not been commercialized.⁹ Aptamer-based assays face significant limitations when it comes to the detection of analytes in undiluted biological fluids.^10,11 Aptamers attain various conformations in a biological fluid; however, a unique folding pattern may favor its efficient binding to a specific target. Single-stranded DNA may form G-quadruplex structures by creating a stack of two or more G-quartets linked by the phosphodiester backbone. Aptamers forming G-quadruplex structures showed high structural stability in different ionic conditions with resistance toward nucleases and higher thermal stability.¹² Furthermore, the selection of oligonucleotide sequences in the presence of a biological matrix helps in the development of aptasensors suitable for biological applications.¹³ It is possible due to the enthalpy-driven selection of aptamers in the presence of a biological matrix during the selection process. It leads to the formation of abundant specific bonds between the ligand and target, compared to an entropy-driven ligand with a similar Gibbs-free-energy change.¹⁴ Other strategies to modify aptamers by chemical modifications of the ribose moiety, circularization, 3′ capping, locked nucleic acid, and so forth, have been used to improve target recognition in the biological fluid, but these changes may compromise the target binding affinity.^15,16

Several aptasensors have been developed to detect progesterone in buffer or in diluted serum samples, but none of these has been validated in undiluted serum or milk samples (Supporting Information Table S1). Dilution of biological samples often compromises assay sensitivity. In the present work, the SELEX protocol has been designed in a way to select a stable G-quadruplex forming progesterone aptamer to detect in undiluted biological samples through the development of an impedimetric biosensor.

Experimental Section

Modified SELEX Using Complex Matrices

The selection of aptamers against progesterone was performed as described elsewhere with certain modifications.¹⁷ Briefly, the ssDNA library was flagged with a biotinylated linker (5′-bio-TCAAGTGGTCATGTACTAGTCAA-3′) by mixing them in 1:2 molar ratio, followed by incubation at 95 °C for 10 min, 55 °C for 1 min, and finally at 25 °C for 1 min. The flagged DNA library was immobilized on streptavidin-coated magnetic beads (400 μg) by incubating at room temperature for 2 h with gentle rotation. The DNA library immobilized on streptavidin-coated magnetic beads was washed four times with a binding buffer (pH 7.4; 1 mM CaCl₂, 2.5 mM KCl, 0.75 mM KH₂PO₄, 0.5 mM MgCl₂·4H₂O, 135 mM NaCl, 8 mM Na₂HPO₄·7H₂O) to remove the unbounded oligonucleotides and incubated with 100 μM progesterone for 15 min at room temperature with mild vortexing. The supernatant containing progesterone–DNA complex was ethanol-precipitated and resuspended in nuclease-free water. The recovered ssDNA was quantified (Nanodrop2000 spectrophotometer; Thermo Fisher Scientific, USA) and amplified through PCR using forward (5′-TAGGGAAGAGAAGGACATATGAT-3′) and biotinylated reverse (5′-bio-TCAAGTGGTCATGTACTAGTCAA-3′) primers under the PCR conditions—initial denaturation at 95 °C for 5 min, followed by denaturation (95 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 30 s) with final extension (72 °C, 5 min) for 25 cycles. The amplicon was incubated with streptavidin-coated magnetic beads and denatured by alkali treatment (125 mM NaOH) at 37 °C for 10 min, followed by removal of the biotinylated strand to generate the secondary ssDNA library. The secondary library generated at each cycle was used in the subsequent cycle of SELEX. The recovery rate after completion of each cycle was estimated by using the formula [(recovered ssDNA/input ssDNA) × 100]. The counter-selection of the aptamer pool was performed using a structurally similar molecule (β-estradiol; 100 μM) as well as with the biological matrix (milk and serum). The oligonucleotides immobilized on beads were recovered during counter-selection to generate a secondary library. Each step of counter-selection was followed by two cycles of SELEX using progesterone as a target. The enriched pool of oligonucleotides obtained after the 19th cycle was cloned and sequenced, as discussed in Supporting Information Section S3 (Supporting Information Figure S2).

Screening of G-quadruplex Forming Oligonucleotides Binding with Progesterone

Oligonucleotide sequences obtained after sequencing were analyzed through “QGRS Mapper” (http://bioinformatics.ramapo.edu/QGRS/analyze.php), a web tool to analyze their ability to form G-quadruplex structures. Further, the secondary structure of G-quadruplex forming oligonucleotides was predicted by circular dichroism (CD) spectroscopy. The aptamer (1 μM) was dissolved in the binding buffer containing 10 mM KCl, and the spectrum was recorded from 200 to 300 nm wavelength using a 0.2 cm path length cuvette at 25 °C with 100 nm/min scanning speed. An average of three CD spectra was recorded, and the data were normalized with blank.

Aptamer Adsorption–Desorption Assay Using Gold Nanoparticles

Gold nanoparticle-based adsorption–desorption assay was performed to analyze the aptamer-progesterone binding as described elsewhere with certain modifications.¹⁸ Citrate-stabilized gold nanoparticles (10 μL; 20 nm size) were mixed with an optimized aptamer concentration (150 nM) and incubated for 30 min at room temperature. After that, an optimized concentration of sodium chloride (30 mM) was added before the addition of progesterone to the aptamer-adsorbed gold nanoparticle suspension. After 10 min of incubation at room temperature, changes in color and absorbance (A_650/520) were measured to estimate the extent of aptamer desorption from gold nanoparticles upon the addition of progesterone.

Stability of Selected Aptamers in Biological Fluids

Potential progesterone binding aptamers were checked for their stability in undiluted milk and serum. For this, 10 μL (1 μM) of aptamer was incubated in an equal volume of milk and serum for 0 and 60 min at room temperature. After that, the samples were loaded on 3% agarose gel to observe the oligonucleotide integrity upon electrophoresis. The gel band intensity at different time points was quantified using ImageJ software.

GQ5-Progesterone Interaction, Molecular Docking, and Binding Affinity Determination

The binding affinity of the aptamer toward progesterone was estimated by microscale thermophoresis (MST) by following the methods mentioned elsewhere.¹⁹ Briefly, varying concentrations (1:2 serial dilution) of progesterone were added to the aptamer (10 nM; 5′ Cy5 labeled) in MST buffer and loaded in 16 MST grade capillaries. The fluorescence signal of the aptamer–progesterone complex was normalized with an unbound fraction and used to estimate the binding affinity (K_D). The change in fluorescence intensity was plotted as a nonlinear regression curve. To test the selectivity of the aptamer, the same experiment was performed with homologous molecules such as β-estradiol, norethisterone, and ampicillin. The molecular interaction between the aptamer and progesterone was studied by CD spectroscopy and molecular docking (Supporting Information Section S4).

Fabrication of the Impedimetric Aptasensor for Detection of Progesterone

Electrochemical impedance spectroscopy (EIS) was performed using a potentiostat three-electrode system to develop progesterone aptasensor following the methodologies described by Zhang et al. (2018)²⁰ with certain modifications. The 5 mM K₃Fe(CN)₆ and 25 mM KNO₃ electrolytic solution was applied as redox reporters to measure the electrochemical signal at the electrode surface. For EIS, each measurement was swept from 10 kHz to 0.1 Hz at a bias potential of 0.3 V with an alternating potential of 10 mV. The parameters in the equivalent circuit such as electrolyte resistance (R_e), charge-transfer resistance (R_ct), and constant phase element were determined from the model circuit using the software PSTrace 5.9.

The optimum aptamer coating concentration, coating time, and the time required for aptamer–target binding were analyzed by recording the impedance signal. The specificity of the electrochemical progesterone sensor was analyzed in a buffer spiked with progesterone, norethisterone, ampicillin, and estradiol. The measurement of impedance at varying progesterone concentrations in spiked buffer, milk, and serum was used to estimate the limit of detection (LOD) for the aptasensor. From the Nyquist plot, the R_ct of each sample was calculated by using the equation [R_ct = (R – R₀)/R₀], where R is the impedance at each concentration of progesterone, while R₀ is the impedance of the blank. The R_ct values for each concentration of progesterone were fitted into a linear regression plot to calculate the LOD using the equation [LOD = (3 × SD)/slope of blank].

Electrochemical Impedance Measurement of Progesterone in the Biological Fluids

For the estimation of progesterone in a biological sample (milk and serum), initially, the impedance response of the aptamer-coated gold electrode was recorded. It was followed by dipping the electrode in the biological sample (milk/serum) for 20 min and washing with deionized water before immersing in the electrolytic solution. The R_ct values at varying progesterone concentrations in spiked milk and serum samples were measured to calculate the LOD. To validate the aptasensor, random milk and serum were collected from the large animal facility at the National Institute of Animal Biotechnology, Hyderabad, India, and their progesterone concentration was estimated using the aptasensor. The result was compared with the standard progesterone—ELISA performed following the manufacturer’s instructions (KINESISDx Los Angeles, USA).

Reproducibility and Stability of the Aptasensor

Regeneration of the aptasensor was carried out by incubating the aptamer-coated electrode in a regeneration buffer (40 mM Tris–HCl, 10 mM EDTA, and 0.02% Tween 20) for 15 min at room temperature, as described by Pandey et al. (2017),²¹ with certain modification. It was followed by the measurement of impedance to ensure complete regeneration. The regeneration followed by the detection of progesterone in the spiked sample was performed several times to analyze the coated electrode’s reusability for progesterone detection. Storage stability of aptamer-coated gold electrode at 4 °C was checked by the daily measurement of impedance in electrolytic solution for 10 days.

Statistical Analysis

All the statistical calculations (mean, RSD, regression analyses) were performed using Graph pad prism 5.0 software. The repeatability of the experiment was calculated as the relative standard deviation percentage (RSD %) for three replicates at three different concentrations. κ value was calculated by using the following derivations

Results and Discussion

Selection of Progesterone—Specific Aptamer through Modified Bead-Based SELEX to Overcome the Matrix Effect

The structural and functional integrity of nucleic acids greatly varies with changes in buffer conditions and ionic strength.²² Because of this, the aptamers selected in a specific buffer work well to recognize their target in that buffer condition. Therefore, an aptamer selected against a target in the buffer may not work universally in the biological fluid for bioassay development.^23,24 For the selection of the aptamer against small molecules like progesterone, immobilization of DNA library rather than target molecules on the solid support was preferred to keep unique functional groups accessible for interaction.²⁵ Therefore, the DNA library was immobilized on streptavidin-coated magnetic beads using a biotin-labeled nucleotide linker. During the SELEX process, a constant yield of recovered DNA in the subsequent cycles was taken as an indication of maximum recovery of progesterone-specific oligonucleotides from the secondary library pool (Supporting Information Figure S1). Counter-selection was performed at this stage to leave behind the non-specific oligonucleotides from the secondary library pool. If we use milk/serum as a medium for the selection of the aptamer, then we will end up with the aptamer selection not only against progesterone but also for complex matrix components of milk and serum. Progesterone dissolved in the buffer was used for SELEX to identify unique DNA sequences that can bind with it. The use of milk and serum as counter targets was to ensure that the enriched pool of potential aptamers do not show cross specificity with components of milk/serum and must have stability against the nucleases present in the milk/serum matrix. A striking decrease in the DNA yield after the counter-selection step might be due to the removal of cross-specific and unstable aptamers (Supporting Information Figure S1). The introduction of two rounds of progesterone SELEX following each counter-selection step was to ensure enrichment of progesterone-specific oligonucleotides. The nucleotide pool obtained at the end of the 19th cycle of SELEX was ligated in the cloning vector and transformed into Escherichia coli. The positive transformants were screened for the presence of the insert in the plasmid DNA upon restriction digestion (Supporting Information Figure S2). Upon sequencing, 241 sequences were obtained, which were further screened through a systematic approach involving in silico analysis, followed by analytical methods, to identify progesterone-specific G quadruplex forming aptamers.

Three G-quadruplex Forming Aptamers Showed Specific Binding with Progesterone

G-quadruplex structures are stabilized by intramolecular or intermolecular interactions, parallel or antiparallel G-rich regions, nucleotide number, and sequence variation in the loop region. It may be due to the higher (twice) negative charge density in G-quadruplex DNA per unit length compared to duplex DNA which facilitates electrostatic and hydrophobic interactions critical for robust and specific binding to the target.²⁶ In silico analysis of 241 aptamers using the QGRS mapper tool led to the identification of 11 G4-forming sequences (Supporting Information Table S1). Such oligonucleotide sequences were further confirmed for the formation of the G4-structure through CD spectroscopy. We observed characteristic parallel G4 spectra with a negative and positive peak at ∼240 and ∼270 nm, respectively (Supporting Information Figure S3).²⁷ However, out of 11, only 3 (GQ3, GQ5, and GQ10) showed a significant (p < 0.05) concentration-dependent binding with progesterone that was evident with the change in color of gold nanoparticles from red to purple (Figure 1A,B). The formation of a blue-colored aggregate in the presence of progesterone was due to the high-binding affinity of progesterone with aptamers, leading to its detachment from the surface of the gold nanoparticle. No change in color of gold nanoparticles was observed when the aptamer was not specific to progesterone (GQ1, GQ2, and GQ8) with the increase in progesterone concentration (Figure 1B). Gold nanoparticle adsorption–desorption assay has been used to estimate the target recognition by the aptamers in buffer.²⁸ However, such an experiment could not be performed in the presence of any kind of biological fluid due to the instability of the gold–aptamer complex. Therefore, the aptamers screened through this assay in buffer were validated for their stability in the biological fluid.

Figure 1.

Screening of aptamers for binding with progesterone and stability in biological fluids. (A) Schematic illustration of aptamer adsorption–desorption assay using gold nanoparticles. (B) Aggregation pattern of gold nanoparticles upon interaction with G-quadruplex aptamers under the increasing concentration of progesterone. The data have been presented as mean ± SD of the A₆₅₀/₅₂₀ from three independent experiments performed in duplicates; (C,D) aptamers were incubated in milk and serum for 0 (immediately after mixing) and 60 min and run onto the agarose gel to observe their integrity. (E,F) Band intensity (mean ± SD) from three independent experiments has been plotted with increasing time of incubation of the aptamer in milk and serum. GQ5 showed specific and high-affinity binding with progesterone.

GQ5 Aptamer Recognized Progesterone with Extended Stability in Biological Fluids

Comparative stability of progesterone-specific aptamers was evaluated in both milk and serum. The samples were loaded onto the agarose gel just after mixing with milk or serum (0 min) and after 60 min of incubation (60 min; Figure 1C–F). The data suggest that GQ3 and P4G11 aptamers got instantly degraded upon exposure to the milk/serum. GQ5 showed about 20% less integrity in milk but was fairly stable in serum (Figure 1C–F). The progesterone aptamer (P4G11 and P4G13) that was used to sense progesterone in diluted serum showed no stability in undiluted milk and serum (Figure 1C–F).¹⁸ However, these were having good stability when incubated with a diluted (1:20) serum sample (Supporting Information Figure S5).

The specificity and binding affinity of GQ5 aptamer with progesterone were analyzed using MST, which measures the molecular movement of the aptamer–target complex through a temperature gradient. A concentration-dependent change in fluorescence signal may be attributed to the alteration in one or more thermophoretic properties like charge, size, and hydration shell upon binding with progesterone (Figure 2A). GQ5 aptamer showed specific binding to progesterone, with a binding affinity (K_D) of 5.29 ± 2.9 nM. However, it did not interact with other molecules like β-estradiol, norethisterone, and ampicillin (Figure 2B–E). The K_D of GQ5 aptamer toward progesterone was found to be the lowest as compared to other progesterone aptamers. GQ5 aptamer adopted the most favorable minimum energy conformation as a stem loop-like structure stabilized by G-quadruplex formed at the junction of loop and stem region with the involvement of G31, G32, G39, and G40 nucleotides. We also noted a concentration-dependent change in the ellipticity of GQ5 aptamer upon incubation with increasing progesterone concentrations (Figure 2G). Progesterone itself does not show any optical activity (Supporting Information Figure S9). The observed change in ellipticity upon the addition of progesterone does not signify a conformational change in the aptamer. However, a concentration-dependent increase and decrease of peak ellipticity at ∼240 and ∼270 nm signifies the probable interaction of progesterone with the backbone of the aptamer as observed in progesterone docked aptamer structure (Figure 2F).

Figure 2.

Biophysical characterization of GQ5 aptamer upon interaction with progesterone. (A) Schematic illustration of the basic principle of microscale thermophoresis (MST) showing the change in the MST traces upon ligand–target interactions. The binding of GQ5 aptamer was determined at increasing concentrations of progesterone (B), estradiol (C), norethisterone (D), and ampicillin (E) by plotting the normalized fluorescence intensity of ligand-bound fraction of the aptamer with the ligand concentration. Nonlinear regression plot has been used to calculate the binding affinity of the ligand with the aptamer. (F) Molecular docking of GQ5 with progesterone. (G) GQ5 was incubated with increasing concentrations of progesterone to record the change in ellipticity. The data have been plotted as the change in ellipticity with an increase in the concentration of progesterone. Analytical performance of the electrochemical aptasensor for progesterone detection.

Through molecular docking, it was predicted that the carbonyl group of progesterone forms H-bond with thiamine (T19) and adenine (A20) nucleotides of the aptamer (Figure 2F). Although these nucleotides are not part of the G-quadrat structure formation, they spatially came closer due to the formation of the G-quadrat structure. Progesterone docked in the non-G-quadrat region with a binding energy of −9.2 kcal/mol. The binding energies of best-docked compounds normally ranged from −8.0 to −11.71 kcal/mol.²⁹ A high binding affinity of the aptamer with progesterone and stability in milk and serum prompted us to use GQ5 to develop an electrochemical aptasensor.

Impedimetric biosensing is a standard method to develop a biosensor for commercial applications.³⁰ It has been used to detect progesterone using GQ5 aptamer in undiluted biological samples in the quantifiable manner. The gold electrode was used for conjugation of the thiol-modified aptamer as the thiol group has strong chemisorption onto the gold surface through the formation of a self-assembled monolayer.^31,32 Successful immobilization of the aptamer on the working electrode was characterized by the measurement of change in impedance due to interference in the redox mediator, leading to an increase in charge-transfer resistance. At an optimized aptamer concentration (3 μM), coating time (4 h), and aptamer–target incubation time (20 min), a significant increase in impedance was observed upon the addition of progesterone (p < 0.05; Supporting Information Figure S6). However, there was no significant change in impedance signal upon the addition of structurally similar or homologous molecules like estradiol, norethisterone, and ampicillin (Supporting Information Figure S6D). When the electrode was used to estimate progesterone in milk or serum, there was an insignificant increase in impedance signal in unspiked milk or serum. Such fluctuation in impedance used to get stabilized in 10–20 min (Supporting Information Figure S8). Recording of the impedimetric signal of progesterone spiked milk or serum was performed only upon stabilization of the impedimetric signal in unspiked samples. The aptasensor showed a concentration-dependent increase in impedance in the spiked buffer, milk, and serum (Figure 3A–C′). The biosensing of progesterone showed a high level of correlation (R²) and LOD as 0.962 and 0.53 ng/mL, 0.9074 and 0.91 ng/mL, and 0.9206 and 1.9 ng/mL in buffer, milk, and serum, respectively (Figure 3A–C′). Progesterone is present in the biological fluids in the range of 1–10 ng/mL. Therefore, it can be inferred from the detection range of the aptasensor that it will find suitability to detect progesterone in biological and environmental samples. The aptasensor also showed good RSD as 2.12, 1.37, and 4.173% in buffer, milk, and serum, respectively. In buffer, the dynamic range of detection was estimated to be 1–50 ng/mL, while in milk and serum, it was 1–30 ng/mL, respectively. A decrease in the dynamic range of detection and the LOD in biological fluids as compared to buffer might be due to the nonspecific adsorption of matrix components onto the electrode.³³ However, this was minimized by blocking the electrode with 10 mM 6-mercaptohexanol. This is the first progesterone aptasensor that can detect it in unprocessed and undiluted milk and serum. The incomplete and partial distortion of the impedance plots in milk and serum samples occurs due to the presence of interfering molecules in the complex biological matrix that creates an interfering layer on the electrode interface. Such kind of change in impedimetric signal was also noted for detection of cancerous cells and bisphenol A.³⁴⁻³⁶ The detection method has been optimized in such a way that the assay sensitivity covers the progesterone concentration at the biological range without introducing several variables during the commercial production of the electrode surface. The methods reported in the previous manuscript used differential pulse voltammetry for progesterone detection in diluted serum.³⁷ We observed that the progesterone aptamers (P4G11 and P4G13) from the previous report⁹ could not detect it in undiluted serum (Supporting Information Figure S7). It was due to the aptamer’s instability in the serum (Figure 1). Therefore, the use of GQ5 to develop impedimetric sensors might find utility for clinical applications. Upscaling of such kind of sensor will be easier for commercial production.

Figure 3.

Electrochemical aptasensor for progesterone detection in buffer, milk, and serum: (A–C) Nyquist plots of EIS measurements with increasing concentrations of progesterone, plotted for detection of progesterone spiked in buffer (A), milk (B), and serum (C). The charge-transfer resistance (R_ct) was fitted against the concentrations of progesterone to plot a linear graph for LOD calculation in buffer (A′), milk (B′), and serum (C′); (D) regeneration capability of the aptasensor was analyzed by plotting the R_ct against various attempts for regeneration of the electrode; (E) R_ct of three aptamer-coated electrodes were measured to ensure uniform and reproducible coating at different gold electrodes under similar conditions; (F) R_ct of the aptamer-coated electrode was measured till 10 days to check its stability. Data are presented as mean ± SD of three independent experiments.

Comparative Efficacy of the Aptasensor with ELISA to Detect Progesterone in the Complex Matrix

The milk and serum samples were obtained from the cow housed at the large animal facility of the National Institute of Animal Biotechnology, India. The milk/serum sample used for the spiking of progesterone was first tested for its presence by ELISA. The sample in which progesterone was undetectable was used for spiking with varying concentrations of progesterone. It was done to avoid any interference in the experiment from the background progesterone present in the biological fluid. Milk and serum samples (n = 9) were analyzed through the aptasensor and ELISA using a standard calibration plot for both assays. A high level of agreement (κ value > 0.9) between the proposed method (aptasensor) and ELISA was observed, indicating its applicability for clinical evaluation (Table 1).

Table 1. Comparative Efficiency of the Aptasensor to Detect Progesterone as Compared to ELISA.

	milk			serum
sr. no.	ELISA (ng/mL)	aptasensor (ng/mL)	sr. no.	ELISA (ng/mL)	aptasensor (ng/mL)
1	2.98	2.55	1	17.57	17.66
2	2.66	3.95	2	2.91	1.82
3	4.41	6.84	3	3.4	3.27
4	6.03	8.78	4	6.1	9.26
5	2.99	4.94	5	2.83	3.22
6	2.88	2.66	6	2.19	3.81
7	4.26	5.99	7	7.63	10.81
8	2.6	2.98	8	4.09	3.74
9	3.44	5.38	9	4.23	7

Regeneration, Precision, and Stability of the Aptasensor

Regeneration of the aptasensor is a very important attribute to enhance the affordability of the sensor. The aptasensor could be regenerated thrice without significant loss in progesterone detection efficiency (Figure 3D). Further, the electrode-to-electrode variation in progesterone detection was checked using three electrodes independently coated with the aptamer under similar conditions. Impedance response of three independent aptamer-coated electrodes showed an RSD of 8.31%, which demonstrate the high precision of the developed aptasensor (Figure 3E). The long-term stability of the aptasensor is a key factor in practical application and commercialization. The aptamer-coated electrode showed less than 10% loss in functional activity upon storage at 4 °C for up to 10 days (Figure 3F).

Conclusions

Ding et al. (2021)¹³ had used bead-based SELEX to select aptamers against sarafloxacin, where they reported the use of milk as a counter-target which improved the development of a fluorescence-based aptasensor. In the present article, we have used milk as well as serum as counter-targets to ensure that the enriched pool of potential aptamers do not show cross specificity with components of milk/serum and must have stability against the nucleases present in the milk/serum matrix. Additionally, rather than target immobilization onto magnetic beads, we immobilized the oligonucleotide library onto the magnetic beads to ensure better target recognition by an aptamer. Thus, in our method of SELEX, the target is free and all the unique functional groups are available for interaction with the aptamer, whereas if the target is immobilized on magnetic beads, all the regions might not be available for aptamer binding. During post-SELEX, the selection of G-quadruplex forming aptamers was preferred due to their inherent higher stability. Further, these aptamers were also checked for stability against the nucleases present in the biological fluids. Therefore, we have optimized the SELEX protocol to select aptamers with high affinity and stability against small molecules (progesterone). The aptamer GQ5 showed extended stability as well as progesterone binding ability in biological fluids. Efficient target recognition by the aptamer in undiluted milk and serum sample may be attributed due to the inclusion of the biological fluid for the counter-selection of aptamers during SELEX. The aptasensor was able to detect the progesterone in undiluted biological fluids within the physiological or pathological limits. Therefore, it can be used in clinical practice as an alternative to ELISA which is expensive and time-consuming.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00185.

• Reagents; instrumentation; aptamer cloning and sequencing; and molecular docking (PDF)

The authors declare no competing financial interest.