2024 Volume 92 Issue 2 Pages 022019
In the present study, a progesterone (P4) biosensor was developed to detect cattle estrus. Thionin, which has been reported to oxidize steroid hormones, was immobilized on an electrode via 10-carboxy-1-decanethiol with the aim of continuous measurement of P4 in the cattle body. On the screen-printed electrode, Au nanoparticles were electrodeposited on the surface of the carbon electrode to increase surface area of electrode. Finally, the thionin-modified electrode surface was covered with Nafion™. As a result, the influence of contaminants (BSA, l-ascorbic acid) was avoided. The detection range of the prepared sensor for P4 was 1 nM (= n mol/l)–20 nM. When bovine plasma was used as a biological sample, it was confirmed that the current response in i-t measurement increased due to the addition of P4. The fabricated biosensor was able to detect P4 for 4 days. It is expected that the P4 biosensor used in the present study will enable accurate understanding of cattle estrus.
Currently, there is a shortage of Japanese black meat cattle in Japan, and the price has nearly doubled in the past 10 years.1 Therefore, there is a need to increase the population of Japanese black cattle. Artificial insemination using fertilized egg transfer, which has a higher conception rate than natural mating, accounts for the majority, but the problem is that the conception rate is low at about 50 %.2 There are two causes for this: individual factors of the cattle and human factors, with deficiencies in the human management system accounting for 90 %.3 An individual factor in cattle is the existence of infertile cattle who have difficulty conceiving. Human factors include the difficulty in distinguishing between cattle in estrus and non-estrus due to individual differences in signs of estrus, and the difficulty in visually monitoring cattle estrus due to the aging of producer and farm scale up.2 The biggest human factor is that producers perform artificial insemination at times other than the optimal fertilization period. The ideal fertilization period for artificial insemination is reported to be 8 h to 12 h after the onset of estrus.4 For this reason, accurately understanding the estrus period of cattle and clarifying the optimal fertilization period for each individual will increase the success rate of fertilized egg transfer and lead to an increase in the number of cattle.
It has been reported that during cattle estrus, an increase in body temperature, a decrease in the pH of the cattle’s cervical mucus, and an increase in electrical conductivity occur.5 Cervical-mounted acceleration sensors have been developed to monitor the riding behavior observed during estrus, and vaginal thermometers have been developed to detect changes in vaginal temperature during estrus.6–8 However, it is difficult to accurately monitor estrus due to the high incidence of false positives due to the difficulty of maintaining the sensor in the proper sensing position, fluctuations in environmental temperature, and disease-related hyperthermia. In addition, sensors using pedometers have been developed focusing on the increase in the number of steps taken during estrus.6,9,10 Furthermore, sensors that utilize changes in the electrical conductivity and pH of cattle cervical mucus during estrus were also investigated.11,12
Therefore, in the present study, we focused on steroid-based sex hormones as the most accurate indicator of estrus. Estrus is induced by sex hormones such as estradiol (E2) and progesterone (P4), so if sex hormone concentrations can be monitored, it is possible to predict upcoming estrus in advance. It has been reported that E2 concentration in blood gradually increases (10 pM (= mol/l) to 30 pM) and P4 concentration in blood decreases (15 nM to 1 nM) starting 96 h before estrus.13 From this, estrus and sex hormones are closely related, and understanding sex hormone concentrations leads to the detection of estrus.
Enzyme-Linked Immuno Sorbent Assay (ELISA) is commonly used to measure the concentration of steroid sex hormones that exist in extremely small amounts in living organisms.14 However, since ELISA kits are disposable, it is difficult to apply them to continuous measurements. In addition, a quantitative method using high performance liquid chromatogram mass spectrometry (LC-MS/MS) has been reported.15 An electrochemical method is a simple method suitable for continuous measurement. However, the targets of the present study, E2 and P4, have low electrochemical activity, making it difficult to measure them using electrochemical methods.
For the purpose of quantifying E2 contained in urine, an electrochemical enzyme biosensor using thionin as a mediator was constructed by Eloy et al.16 Thionin oxidized E2, and E2 was quantified by detecting the current when the reduced thionin was oxidized at the electrode. Furthermore, laccase, an oxidoreductase, was immobilized on the electrode to promote the oxidation of thionine that was not oxidized at the electrode, resulting in highly sensitive E2 quantification.
Our preliminary experiments showed that thionine catalyzes the oxidation of P4 as well as E2. Additionally, the concentration of P4 in the blood is 1000 times higher than E2. Therefore, in the present study, we aimed to construct a biosensor that can continuously measure P4 in vivo over a long period of time. Miniaturization of sensors is essential to reduce the burden on cattle. The electrode used was a screen-printed electrode (SPE) (4 × 12.5 mm) in which the working electrode, counter electrode, and reference electrode were integrated on a single substrate. As shown in Fig. 1, a modified electrode with thionin immobilized on SPE was constructed. In order to increase the amount of modified-thionin aimed at improving the sensitivity of the sensor by increasing the current response, the electrode surface area was expanded by electrodepositing AuNPs on the electrode surface. Thionin was modified into AuNPs electrodeposited on the electrode surface via 10-carboxy-1-decanethiol (10-CDT). Arvand et al. proposed that the allylic hydrogen of P4 is oxidized in the oxidation of P4 by graphene quantum dots.17 Therefore, as shown in Scheme 1, it is thought that the allylic hydrogen of P4 is also oxidized in the oxidation of P4 by thionin in the present study. The electrodes deposited with AuNPs were evaluated by scanning electron microscopy (SEM). The fabricated P4 biosensor was also evaluated for selectivity and long-term stability, and finally, the current response when bovine plasma was used as a biological sample was also evaluated.
Schematic image in electrochemical detection of P4 and P4 biosensor electrode configuration.
Schematic display of the oxidation mechanism of P4 by thionin.
Thionin, HAuCl4 was purchased from FUJIFILM Wako Pure Chemical Corporation. A 10-carboxy-1-decanethiol (10-CDT) was obtained from DOJINDO LABORATORIES. Carbon screen printed electrode (SPE) (TG-1N, d = 3.2 mm) was purchased from BioDevice Technology, Ltd. Nafion was obtained from Merck KGaA. Bovine plasma was provided by Fukui prefecture animal experiment station.
2.2 Electrochemical measurementsCyclic voltammetry (CV) and i-t measurements were performed using a potentiostat (Model 1205B Electrochemical analyzer, BAS Inc.). A three-electrode SPE with carbon as the working and counter electrodes and Ag/AgCl as the reference electrode was used for the measurements. All the potential values are reported with respect to the Ag/AgCl reference electrode. CV was performed at high voltage 0 V, low potential −0.5 V, scan rate 10 mV/s, and sweep segment 2.
2.3 Electrodeposition of Au nanoparticleThe SPE was cleaned by dropping 20 µl of 0.1 M Sodium phosphate buffer (pH 7.0) and performing CV at high potential +1.0 V, low potential −1.0 V, scan speed 0.5 V/s, and sweep segment 50. After washing, the SPE electrodeposited Au nanoparticles (AuNPs) onto the working electrode by dropping 40 µl of 6.0 mM HAuCl4 solution and applying a voltage of −0.3 V for 400 s (AuNPs/SPE).
2.4 Scanning electron microscopyAuNPs/SPE were investigated by scanning electron microscopy (SEM). The AuNPs/SPE were coated with Au/Pd using an ion coater (MSP-1S, VACUUM DEVICE) and observed by SEM at an accelerating voltage of 15 kV (JSM-6390, JEOL).
2.5 Preparation of Nafion/thionin/10-CDT/AuNPs/SPETo prepare AuNPs/SPE, 1 µl of piranha solution was dropped onto the working electrode to remove organic matter. AuNPs/SPE was immersed in 100 µM 10-carboxy-1-decanethiol (10-CDT) dissolved in ethanol for 18 h, and the surface of the AuNPs was modified with 10-CDT via Au-S bonds. Furthermore, after activating the terminal carboxy group of 10-CDT with EDC/NHS, thionin was modified into 10-CDT via amide bond by immersion in a 2 mM thionin solution dissolved in 0.1 M HEPES-NaOH buffer (pH 8.0) for 5 h. Finally, the biosensor was fabricated by dropping 50 µl of 0.05 % Nafion solution onto SPE and air-drying it (Nafion/thionin/10-CDT/AuNPs/SPE).
2.6 Concentration dependence evaluation of Nafion/thionine/10-CDT/AuNPs/SPE on P4 by i-t measurementA 9 µl of 0.1 M sodium phosphate buffer (pH 7.0) was drop on Nafion/thionin/10-CDT/AuNPs/SPE and i-t measurements were performed. The applied voltage was −0.35 V in order to measure thionin-derived oxidation waves, and the current response value was measured when P4 dissolved in DMSO was added for each measurement. Hormone solutions were added sequentially to final concentrations of 1 nM, 5 nM, 10 nM, 15 nM, and 20 nM, respectively.
2.7 Selectivity evaluation of Nafion/thionin/10-CDT/AuNPs/SPESince bovine plasma contains many impurities such as amino acids, sugars, and metabolites, the selectivity of Nafion/thionin/10-CDT/AuNPs/SPE was evaluated by comparing with the response current to interfering substances. The applied voltage was set to −0.35 V. The response current when the P4 solution was added to 0.1 M sodium phosphate buffer (pH 7.0) to a final concentration of 20 nM and the response current when an interference substance was added were measured. The interfering substances selected were BSA (0.1 mg/ml) and l-ascorbic acid (final conc.: 2 µM). BSA and L-ascorbic acid are present in large amounts in biological samples and affect electrode reactions, so they are commonly used as interfering substance models to evaluate the selectivity of biosensors.
2.8 Long-term stability evaluation of Nafion/thionin/10-CDT/AuNPs/SPELong-term stability of Nafion/thionin/10-CDT/AuNPs/SPE was evaluated by i-t measurement. The applied voltage was set at −0.35 V, and a 1 nM (final conc.) P4 solution was measured. After 900 s of voltage application, the P4 solution was dropped, and voltage was applied again for 900 s. Then, the average current response value after 800 to 900 s at each P4 concentration was calculated, and the difference was taken as Δ current. Tests were conducted every other day, and three electrodes were tested independently. In addition, the electrodes used for evaluation were stored at 4 °C under a N2 atmosphere, shielded from light.
2.9 i-t measurement using bovine plasma as a biological sampleThe performance of the P4 biosensor was investigated by i-t measurement of Nafion/Thionin/10-CDT/AuNPs/SPE when bovine plasma was used as a biological sample. The applied voltage was set at −0.35 V. Measurement was started by dropping 20 µl of bovine plasma was dropped onto the sensor. After 1500 sec, P4 solution was added to increase the P4 concentration in the sample by 10 nM, and the response current was measured.
Figures 2a and 2b show SEM images before and after electrodeposition onto the working electrode on SPE. Before electrodeposition as shown Fig. 2a, the carbon electrode surface was exposed. On the other hand, after electrodeposition as shown Fig. 2b, the surface was covered with nanoparticles, and the particle size was approximately 200 nm. This is because the electrodeposition modified the working electrode surface with AuNPs, increasing the electrode area. To calculate the electrochemical electrode surface area, CV was performed on AuNPs/SPE using an electrolyte containing 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide (Data not shown). From the CV results and calculated using the Randles-Sevciks equation, the surface area of the working electrode of AuNPs/SPE was 1.6 times larger than that of the bare electrode.
SEM images (10000× magnification, scale bar = 1 µm) of the working electrode (a) before and (b) after electrodeposition of AuNPs.
Nafion/thionin/10-CDT/AuNPs/SPE was evaluated electrochemical characteristic by CV. Figure 3 shows the CVgrams of 10-CDT/AuNPs/SPE (thionin unmodified) and Fig. 4 shows the CVgrams of Nafion/thionin/10-CDT/AuNPs/SPE (thionin modified). In Fig. 3, a reduction peak was observed around −0.3 V. The working electrode surface of SPE is mostly covered with Au nanoparticles, and amorphous carbon is exposed in some areas. Therefore, this reduction peak is a peak typically observed in CV using an amorphous carbon electrode in phosphate buffer.18 In addition, the reduction peak around −0.3 V shifted to the negative side with the addition of P4. This is due to the addition of DMSO, which was used as a solvent for P4. However, as shown in Fig. 4, no peak shift due to the addition of P4 was observed in the Nafion/thionin/10-CDT/AuNPs/SPE, so DMSO does not have a large effect on electrochemical measurements in Nafion/thionin/10-CDT/AuNPs/SPE. In Fig. 4, the oxidation peaks at −0.18 V and −0.37 V and the reduction peak at −0.27 V originate from the redox of thionin. This result indicates the successful immobilization of thionin onto the electrode surface. In addition, when thionine is modified to 10-CDT, some thionine adsorbs to the carbon surface, covering the carbon surface. Therefore, the reduction peak around −0.3 V observed in CV using a thionin-modified electrode is a reduction peak derived from thionin. Furthermore, the addition of P4 increased the oxidation current around −0.37 V. This is because the amount of reduced thionin on the electrode increased due to the oxidation of P4 by thionin. From the CV results, the applied voltage in i-t measurements was determined to be −0.35 V.
CVgrams of 10-CDT/AuNPs/SPE without thionin using 0.1 M sodium phosphate buffer (pH 7.0) as electrolyte without P4 (dotted line) and with 20 nM P4 (solid line).
CVgrams of Nafion/thionine/10-CDT/AuNPs/SPE using 0.1 M sodium phosphate buffer (pH 7.0) as electrolyte without P4 (dotted line) and with 20 nM P4 (solid line).
The concentration dependence of Nafion/thionin/10-CDT/AuNPs/SPE for P4 was evaluated by i-t measurement. Figure 5a shows the results of i-t measurements using Nafion/thionin/10-CDT/AuNPs/SPE. An increases in the response current, i, were confirmed with the addition of P4, as indicated by the arrow. Figure 5b shows the semi-log plot of current value versus P4 concentration in Fig. 5a. Δi is a value calculated by subtracting i at 0 nM p4 from i before addition of each P4 solution. The biosensor showed good linearity in the range of 1 nM–20 nM. However, since the change in response current is small, it is necessary to improve the response current.
(a) i-t curve of Nafion/thionin/10-CDT/AuNPs/SPE using 0.1 M sodium phosphate buffer (pH 7.0) as electrolyte. (b) Semi-log plot of current value versus P4 concentration.
The selectivity of the biosensor was evaluated by comparing the response current in i-t measurements with Nafion/thionin/10-CDT/AuNPs/SPE to interferences. Figure 6 shows the difference in response current values before and after the addition of each impurity. The Δi when adding P4, l-ascorbic acid, and BSA were 61 nA, −7 nA, and −3 nA, respectively. The response current increased when P4 was added. On the other hand, no large Δi was observed. This is because Nafion prevented l-ascorbic acid, which has a negative charge, and BSA, which has a large size, from approaching the electrode. Therefore, the use of Nafion makes it possible to measure P4 in biological samples using this biosensor.
Current response of Nafion/thionin/10-CDT/AuNPs/SPE for each interfering substance. P4: 20 nM, l-ascorbic acid: 2 µM, BSA: 0.1 mg/ml.
The long-term stability of the P4 biosensor was electrochemically evaluated by i-t measurements. Figure 7 shows the results of long-term stability evaluation. The Δi of the P4 biosensor did not decrease until the third day. At day 5, the Δi decreased to approximately 15 % and showed no response to P4 at day 7. In contrast, with Nafion unmodified electrodes, the response current was maintained after 2 weeks (data not shown). This may be because the large negative charge of Nafion attracts positively charged oxidized thionin, reducing its reactivity with P4 and causing thionin decomposition. It is necessary to select a material to replace Nafion that prevents contaminants, or to introduce a method to suppress the effect of Nafion on thionin. The estrus cycle of cattle is 21 days, and this P4 biosensor aims to provide continuous measurement in the future. Therefore, further improvements are required regarding the long-term stability of the biosensor.
Long-term stability of the P4 biosensor.
Finally, i-t measurements were performed using bovine plasma as a biological sample. Figure 8 shows the results of i-t measurement using bovine plasma as a biological sample. When P4 solution was added onto the electrode, the i increased. The increase in response current value due to the addition of P4 solution was 116 nA. From this result, this P4 biosensor can respond to changes in P4 concentration in bovine plasma. In the measurements using actual samples, a P4 solution was added so that the P4 concentration in the sample increased by 10 nM. However, in the calibration curve in Fig. 5, the Δi corresponding to a concentration of 10 nM is approximately 200 nA, which is large compared to the Δi in measurements using actual samples. This is because the electrode reaction is slightly affected by impurities contained in bovine plasma that are unavoidable with Nafion. In the future, it will be necessary to compare the response currents when using estrus plasma and non-estrus plasma as samples.
i-t curve of P4 biosensor using bovine plasma as a biological sample.
In the present study, we constructed an electrochemical P4 sensor based on the oxidizing ability of thionin to P4 for the purpose of detecting estrus in cattle. Thionin was modified to AuNPs electrodeposited on the electrode surface via 10-CDT, and finally the electrode surface was covered with Nafion. This P4 biosensor showed good linearity in the P4 concentration range of 1 nM–20 nM. In the selectivity evaluation, this biosensor did not show a response current to l-ascorbic acid and BSA by covering with Nafion. As a result of long-term stability evaluation, this P4 biosensor showed a response current to P4 for 5 days. When bovine plasma was used as a biological sample, this P4 sensor showed a response current to changes in P4 concentration. The detection range of this P4 biosensor sufficiently covers changes in P4 concentration during estrus in cattle, and it is expected that it will enable easy and accurate understanding of the time of estrus in cattle. In the future, we will provide sensors to monitor cattle estrus in real time.
Eiichiro Takamura: Conceptualization (Lead), Funding acquisition (Lead), Writing – original draft (Lead)
Taiga Yashiki: Validation (Equal)
Kyouhei Inada: Resources (Equal)
Kaname Suzuki: Resources (Equal)
Nobuhiro Kawamori: Resources (Equal)
Hiroaki Sakamoto: Writing – review & editing (Equal)
Takenori Satomura: Writing – review & editing (Supporting)
Shin-ichiro Suye: Writing – review & editing (Supporting)
The authors declare no conflict of interest in the manuscript.
H. Sakamoto: ECSJ Active Member
