Highly sensitive biosensors for real-time monitoring of histamine at acupoint PC6 in rats based on graphene-modified acupuncture needles

Abstract

Acupoints are the local initial response sites of acupuncture therapeutic effects. As a biomarker, histamine is released into the acupoint region and plays its role concurrently as acupuncture needles are inserted into acupoints. Hence, real-time monitoring of histamine at acupoints is important to elucidate the effectiveness of the acupoint-activation process in acupuncture. Therefore, we developed highly sensitive acupuncture/Au particles/graphene biosensors by electrodeposition, brushing, and annealing methods based on bare acupuncture needles. We achieved a histamine detection limit of approximately 4.352 (±3.419) × 10⁻¹² mol L⁻¹ and good sensitivity of approximately 6.296 (±3.873) μA μM⁻¹, with satisfactory specificity, repeatability, and stability in vitro, rendering them more competitive and suitable for real-time monitoring in vivo without causing additional damage. Subsequently, we conducted real-time histamine monitoring at non-acupoint and acupoint PC6 in rats, respectively. Our results showed minimal changes at the non-acupoint, whereas a trend of initial increase followed by a decrease was observed at acupoint PC6. The change in histamine concentration at acupoint PC6 reflected its involvement in the acupoint-activation procedure. Moreover, its peak position at ∼18 min could provide guidance for optimizing needle retaining time for maximum therapeutic effect. This work presents the first real-time in vivo monitoring of histamine at acupoints with high sensitivity and underscores the specificity of histamine release between non-acupoint and acupoint PC6, demonstrating great potential for elucidating the acupoint-activation mechanisms in acupuncture. Additionally, this work expands the application of nanomaterials in the integration of medicine and engineering, which is an important aspect of the future development of materials science.

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1. Introduction

Acupuncture is an integral part of traditional Chinese medicine (TCM) that is well-known as an effective clinical therapy all over the world. As the local initial response sites of acupuncture stimulation, acupoints, which are located at specific anatomical positions throughout the body, play a crucial role in producing clinically significant therapeutic effects. The acupuncture stimulation at acupoints can promote the local release of a series of biochemical substances, such as histamine, 5-HT (serotonin), Ca²⁺, and so on, activating the nerve-endocrine-immunity regulation network in the human body, which results in a wide range of regulation effects. Therefore, the detection and analysis of various biochemical substances in the micro-environment of acupoints promotes our understanding of the acupoints-activation process for acupuncture effectiveness.

As a biogenic amine, histamine is widely present in mast cells at acupoints and is released into the acupoint region during the degranulation of mast cells caused by acupuncture. Early studies have proved that the release of histamine and other substances at relevant sensitized acupoints might directly affect the nerve-endocrine-immunity regulation network.¹ Meanwhile, histamine^2,3 could also relax blood vessels, enhance vascular permeability, and participate in the body's humoral reaction process, which eventually affects the nerve-endocrine-immunity regulation network and promotes clinical efficacy⁴ of acupuncture. Therefore, it is important to identify the real-time changes in histamine concentration at acupoints for recognizing the acupoints-activation process of acupuncture effectiveness.

Various detection methods of histamine have been reported, including thin-layer chromatography (TLC),⁵ gas chromatography,^6,7 capillary electrophoresis,⁸ high-performance liquid chromatography,^9,10 fluorescence method,¹¹ enzyme-linked immunosorbent assay,¹² and so on. These methods are usually utilized for the detection of histamine in fish, while the monitoring of histamine at the acupoints during acupuncture is absent. Moreover, when applied at acupoints, these methods might lack sensitivity and could potentially bring about harm to human bodies or other experimental animals due to their limitations in real-time and in vivo monitoring. In this regard, the real-time, high-sensitivity monitoring of histamine at acupoints remains a significant, but challenging, topic to explore.

In fact, the incorporation of biosensors with acupuncture needles has rapidly developed in recent years because they hold the potential to provide real-time monitoring of in vivo physiological changes during acupuncture procedures, thereby advancing the scientific and quantitative research of acupuncture.^13–15 For example, acupuncture needles are applied as electrochemical electrodes to monitor biological mediators, such as promethazine, chlorpromazine, and epinephrine.^16–19 Guo et al. pioneered the development of calcium ion sensors using acupuncture needles.²⁰ Lin et al. applied acupuncture needles for lactate detection with a polymer modifier for enzyme immobilization.²¹ Tang et al. prepared graphene-modified acupuncture needles for neurotransmitter detection.²² Niu et al. fabricated an acupuncture needle electrode for the sensitive determination of rutin.¹⁹ Yang et al. successfully developed a molecularly imprinted miniature electrochemical biosensor for SARS-CoV-2 spike protein based on Au nanoparticles and reduced graphene oxide-modified acupuncture needles.²³ These biosensors, based on acupuncture needles are reliable and promising for future applications.

Based on this, we aimed to explore the feasibility of developing a needle-type biosensor for highly sensitive and real-time monitoring of histamine at animals’ acupoints without causing additional damage. In this work, we developed a highly sensitive histamine electrochemical biosensor, designated as AN/AuPs/G (where the symbol “/” was used to indicate the direct contact between different layers) based on bare acupuncture needles (ANs). Gold particles (AuPs) and graphene (G) were sequentially, to modify the ANs, where we leveraged the high conductivity, good biocompatibility, and electrocatalytic ability of AuPs^24–26 to enhance the efficiency and reversibility of electron exchange between acupuncture needle surface and graphene interface; G was selected due to its high quality, large lateral sizes, and excellent electronic conductivity as a highly sensitive layer for histamine detection. Through the synergistic effects of AuPs and G, a detection limit of approximately 4.352 (±3.419) × 10⁻¹² mol L⁻¹ and good sensitivity of approximately 6.296 (±3.873) μA μM⁻¹ was achieved in vitro for our AN/AuPs/G electrochemical system; furthermore, we successfully achieved real-time monitoring of histamine concentrations at acupoint PC6 in rats during acupuncture treatment without introducing additional damages, thus facilitating wider applications of the biosensors proposed in this study.

2. Materials and methods

2.1 Materials and reagents

Stainless steel ANs (size 0.25 × 30 mm, Lot no. 23071021) were purchased from Beijing Zhongyan Taihe Medical Instrument Co., Ltd (Beijing, China). Graphene was synthesized utilizing a glue-assisted grinding exfoliation (GAGE) method: In a typical GAGE process, taking the exfoliation of graphite powder by CMC solution as an example, the bulk graphite powder (7 g) and the CMC solution (150 g, the content of CMC is 3 g) were added to a mortar grinder and ground for 9 h (Retsch, RM 200). During the grinding process, the mortar was rotated at a speed of 100 rpm, and the graphite powders were exfoliated into 2D graphene nanosheets in an ambient atmosphere. A homogeneous graphene nanosheets/CMC dispersion was obtained and used directly for further applications.²⁷

Chloroauric acid (HAuCl₄) and potassium ferricyanide (K₃FeC₆N₆) were obtained from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China) and stored in a dark place. Histamine (C₅H₉N₃), anhydrous potassium chloride (KCl), anhydrous calcium chloride (CaCl₂), hydrochloric acid (HCl), sodium hydroxide (NaOH), and phosphate-buffered saline (PBS, 1 mol L⁻¹) were all obtained from Beijing Abfans Technology Co., Ltd (Beijing, China). Among these, PBS acted as the electrolyte, and its PH values were adjusted by adding HCl or NaOH. Histidine (C₆H₉N₃O₂) and putrescine (C₅H₁₄N₂) were purchased from Beijing Bailingwei Technology Co., Ltd (Beijing, China) and Nantong Jingwei Biotechnology Co., Ltd (Nantong, China), respectively. All these reagents and other chemicals employed were of analytical grade. Double distilled water was used throughout this study.

2.2 Apparatus

A CHI 660E electrochemical workstation (Shanghai Chenhua Instrument, Co., Ltd, China) was used to electrodeposit the gold particles. All electrochemical experiments, including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV), and open circuit potential (OCPT) were also performed on it. A conventional three-electrode system was used with the modified electrode as the working electrode, the saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode, respectively. In order to improve the performance and the stability of our AN/AuPs/G biosensor, an annealing treatment was conducted using the OTF-1200X-S Micro-open tube furnace (Sino-us joint venture Hefei Kejing Material Technology Co., Ltd). The biosensor's surface, cross-section, and its responding energy dispersive spectrometer (EDS) were detected by the new field emission scanning electron microscope (SEM, SU8200 series, Hitachi Ltd, Japan).

2.3 Electrochemical measurements

To carry out the electrochemical detection, CV and DPV were utilized in a three-electrode system in 1× PBS solution. CVs were performed at the range of −1–0.5 V at a scan rate of 100 mV s⁻¹.²² The electrochemical parameters for DPVs were: −0.8–0.2 V, step potential, 0.004 V; amplitude, 0.05 V; pulse width, 0.2 s; pulse period, 0.5 s. EIS was employed in 0.1 M KCl (containing 5.0 mM K₃[Fe(CN)₆]). All measurements were performed at room temperature.

3. Results and discussion

3.1 Fabrication of modified AN/AuPs/G electrodes

We started with a bare stainless steel AN as the substrate, upon which a modified AN/AuPs/G electrode was assessed (the whole procedure is summarized in Fig. 1). First, the AN underwent ultrasonic cleaning for 20 min in double-distilled water and absolute ethyl alcohol, respectively, followed by drying with N₂. A layer of AuPs was then deposited onto the tip (1 cm) of the ANs (resulting in AN/AuPs) using a deposition potential of −0.4 V and a deposition time of 50 s in a 2.5 mmol L⁻¹ HAuCl₄ solution maintained at 50 °C in a water bath, as described in the previous literature.¹⁹ Following this, to further remove any potential residual organics within the G and enhance the adhesion of different layers, we applied a graphene slurry evenly onto the surface of AN/AuPs through brushing, after which the electrodes were annealed at 400 °C under the flow of a mixture of hydrogen and argon (with a volume fraction of H₂ 15%) for 1 hour. Finally, the rest of the needle body was covered by an insulated epoxy resin membrane. Upon drying, the final modified electrodes (AN/AuPs/G) were achieved.

Fig. 1 Schematic illustration of the fabrication process of the modified AN/AuPs/G electrodes.

3.2 Characterization of AN/AuPs/G electrodes

As shown in Fig. 2, the morphology and microstructure of the fabricated electrodes at different stages were investigated by SEM. The pristine AN had a smooth and uniform surface with a tip diameter of ∼10 μm (Fig. 2A and D). After electrochemical deposition of AuPs onto the surface of the tip of the AN, we measured the grain size of our Au particles, which revealed that the diameters of the majority of the Au particles ranged from approximately 100 to 200 nm. In addition, several clusters were scattered on the surface that measured no more than 500 nm. The deposition of AuPs on the AN surface enhanced the surface area, providing a roughness interface that was suitable for the modifications that followed. Meanwhile, Au particles could enhance the electrochemical signal and adsorption capacity, thereby improving the detection sensitivity.²⁸ Subsequent G modification was followed by annealing of the AN/AuPs/G at 400 °C under a flow of hydrogen and argon (with a volume fraction of H₂ 15%) to enhance the adhesion between layers and remove the possible residual impurity. As we can see in Fig. 2C and F, the G, nanosheets fully covered the surface of the AN/AuPs, indicating successful modification of both AuPs and G onto the AN/AuPs/G as intended.

Fig. 2 SEM characterization. (A–C) The morphology of different as-prepared electrodes (A: AN; B; AN/AuPs; C: AN/AuPs/G). (D–F) The microstructure of different as-prepared electrodes (D: AN; E; AN/AuPs; F: AN/AuPs/G). (G) The cross-section of the AN/AuPs/G electrodes. (H–I) EDS elemental mapping of the AN/AuPs/G electrodes.

Cross-sectional imaging demonstrated close and conformal adhesion between different layers, facilitated by the flexibility of G and the high-temperature annealing process. The thickness of G was about 10 to tens of μm, as illustrated in Fig. 2G. Thus, the effective surface area of the AN/AuPs/G electrode was approximately 8.37 mm², compared to 7.46 mm² for the bare AN. Furthermore, the energy dispersive spectrometer of the AN/AuPs/G electrode confirmed the presence of Au, C, and Fe, with minimal N, P (Fig. 1H, I and Fig. S1†), indicating the purity of G and the successful preparation of the electrode.

3.3 In vitro detection performance of AN/AuPs/G biosensors

3.3.1 Electrochemical properties of the AN/AuPs/G biosensors. The electrochemical properties of different modified electrodes in a 0.1 M KCl solution (containing 5.0 mM K₃[Fe (CN)₆]) were examined via cyclic voltammetry. This method is effective for monitoring the electron transmission procedure in the modified electrode.²⁹ As we can see in Fig. 3A, the AN biosensor exhibited minimal transmission, indicating weak reactions on the AN electrode. Both the AN/G and AN/AuPs biosensors displayed larger responses, suggesting that both AuPs feature their conductivity and catalytic properties, while G exhibits good sensitivity. These properties effectively enhanced the electrochemical properties of the bare AN electrode. The potential synergy between AuPs and G was anticipated. Indeed, AN/AuPs/G biosensors exhibit quasi-reversible electrochemical reaction processes (with a pair of quasi-symmetric peaks) and a more substantial redox reaction, compared to AN/AuPs or AN/G biosensors, suggesting that the synergistic effect of G and AuPs significantly facilitated the performance of the biosensors. Additionally, we compared the CV curves of the AN/AuPs/G biosensors with those of the AN/AuPs/rGO biosensors that were prepared using conventional methods described in previous literature. As shown in Fig. S7,† the redox peaks of AN/AuPs/G (unannealed) and AN/AuPs/G (annealed) biosensors increased slightly and significantly, respectively, compared to those of the AN/AuPs/rGO biosensors. This suggested that the preparation methods, especially the annealing process emphasized in this study, played a crucial role in enhancing the properties of the AN/AuPs/G biosensors. Furthermore, the electrochemical impedance spectroscopy (EIS) of different electrodes in a 0.1 M KCl solution (containing 5.0 mM K₃[Fe (CN)₆]) were recorded, which is among the most powerful tools for interfacial investigation³⁰ (Fig. S2†). As a result, the EIS of the AN/AuPs/G biosensor displayed a small semicircle at high frequencies, suggesting very low electron-transfer resistance (R_et) because R_et was equal to the diameter of the semicircle of the Nyquist plots.

Fig. 3 (A) CVs on AN, AN/AuPs, AN/G, and AN/AuPs/G biosensors in a 0.1 M KCl solution (containing 5.0 mM K₃[Fe(CN)₆]) with the scan rate of 100 mV s⁻¹. (B) CVs of different scan rates on AN/AuPs/G biosensors. (C) CVs at different pH values (from 5 to 11) on AN/AuPs/G biosensors. (D) Relationship between pH and oxidation peak value. (E) DPV curves of different histamine concentrations (from 0 to 0.3 mg L⁻¹) on AN/AuPs/G biosensors. (F) Linear relationship of peak value and histamine concentrations.

3.3.2 Effect of scan rate and PH on the AN/AuPs/G biosensors. In Fig. 3B, CV curves of the AN/AuPs/G biosensor as it detected histamine (5 mg L⁻¹) in PBS at different scan rates from 20 mV s⁻¹ to 300 mV s⁻¹ are displayed. The shapes of the CV cures remained consistent, with the redox peak increasing with the scan rates, forming linear relationships with the square roots of scan rates. The linear relationship between the oxidation peak value and the square roots of scanning rates: I(A) = 0.463(±0.019)X − 1.585(±0.223), R² = 0.988, conformed with the Randles–Savcik equation, suggesting that the histamine sensing process was primarily diffusion-controlled.^31,32

Solution pH was another crucial factor influencing the performance of the biosensor. We varied the pH of the solution between 5.0 and 11.0 to study its impact on the CV trends of histamine (5 mg L⁻¹) in PBS. It can be seen in Fig. 3C and D that the peak current of the AN/AuPs/G biosensor increased until pH 6.0 and decreased thereafter. pH variations affected the concentration of hydrogen ions in the solution, thereby influencing the reaction rate and charge transfer processes at the electrode surface, leading to a shift in the peak position of the redox reaction. Ultimately, pH 6.0 was identified as the optimal pH for the working buffer and was used in subsequent in vitro experiments.

3.3.3 Performance of AN/AuPs/G biosensors for detecting histamine in vitro. Differential pulse voltammetry (DPV) was employed to establish the calibration curve for histamine determination owing to its superior sensitivity and peak shape. Fig. 3E displays the DPV responses of various histamine concentrations on AN/AuPs/G biosensors under pH 6.0 conditions. We found that the peak value increased gradually with the addition of histamine (a similar trend was observed in the CV response as displayed in Fig. S3†). Linear calibration curves were established in two regimens, ranging from 0–0.06 mg L⁻¹ with a linear regression equation of I₁(A) = 6.964(±0.578)X + 2.353(±0.021) (R² = 0.967) and from 0.07–0.3 mg L⁻¹ with a linear regression equation of I₂(A) = 0.699(±0.066)X + 2.746(±0.011) (R² = 0.965), respectively (Fig. 3F). The lowest detection value was determined to be approximately 4.352 (±3.419) × 10⁻¹² mol L⁻¹ (n = 3), which was calculated from the relationship, LOD = 3.3δ/S, where δ is the standard deviation of three measurements of the blank solution. S denoted the slope of the calibration curve in the first linear range. This result was significantly lower compared to previous findings, which was possibly due to the high conductivity of the modified electrode with a large effective surface area (i.e., ∼8.37 mm²). Additionally, the sensitivity of the AN/AuPs/G biosensors was calculated to be ∼6.296 (±3.873) μA μM⁻¹ (n = 3) using the formula: S = (I₂ − I₁)/(C₂ − C₁), where I and C are the oxidation peak values and the concentration of histamine in the first linear range respectively, suggesting excellent detection properties of our AN/AuPs/G biosensors.

Furthermore, we compared our results with some other previous works for histamine detection, as summarized in Table 1, indicating that our modified AN/AuPs/G biosensor had a superior detection limit compared with other reported sensors. Furthermore, we compared the sensitivity of our AN/AuPs/G biosensors (6.296 ± 3.873 μA μM⁻¹) with those reported in previous literature³⁴ (0.0631 μA μM⁻¹), demonstrating a significantly higher sensitivity of our AN/AuPs/G biosensors. It is worth noting that the previous literature does not provide information regarding sensitivity. Therefore, AN/AuPs/G biosensors present a promising avenue for electrochemical detection of histamine concentrations at acupoints in live animals.

Table 1 Comparison of the analytical parameters of different modified electrodes for histamine analysis

Modified electrode	Linear range (μmol L^−1)	LOD (nmol L^−1)	Sample	Ref.
MIP-AuNPs-GCE	0.002–0.9	1.98	Histamine in KCl solution	33
(GPH/chitosan/CSPE)	0.1–300	25.4	Histamine in PBS solution	34
Cu NCs-modified electrodes	0.010–100	2.5	Histamine in PBS solution	35
SPGEs	45–900	5585.59	Histamine in NaOH solution	36
CeO2/RGO/GCE	2.25–900	1441.44	Histamine in PBS solution	37
CSCNF electrode	0.3–300	100	Histamine in the HBSS (+) solution	38
Magnetic immunochromatographic	9–900	10 811	Histamine in PB solution	39
MIP/SPE	0.001–1	1.765	Histamine in PBS solution	40
Polyurethane-LiClO4 to modify screen-printed electrodes	150–1000	170 000	Histamine in KCl solution	41
ECL sensor	0.1–200	17	Histamine in Tris-HCl solution	42
AN/AuPs/G	0.001–0.3	0.044	Histamine in PBS solution	This work

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3.3.4 Specificity, repeatability, and stability of AN/AuPs/G biosensors. The influence of foreign species was crucial in assessing the performance of biosensors. Therefore, we compared the histamine responses with those of amine substances (histidine, putrescine), as well as acupoint-related substances (K⁺, Ca²⁺), using the CV method (Fig. 4A). All substances maintained the same concentration (refer to Table S1†). It was evident that biosensors with AN/AuPs/G as the electrode demonstrated superior sensing ability, compared to other amine substances and other acupoint-related substances (Fig. 4B).

Fig. 4 (A) Cyclic voltammetry curves of different substances. (B) Oxidation peak value of different substances. (C) Cyclic voltammetry curves of different cycles. (D) Cyclic voltammetry curves after different days.

Repeatability and stability were other essential characteristics of biosensors. To evaluate the repeatability of the AN/AuPs/G biosensors, two hundred successive measurements were conducted in a 5 mg L⁻¹ histamine solution on one AN/AuPs/G biosensor. The resulting current response remained at 92.486% (±5.183%; n = 5) (Fig. 4C) of the initial value, demonstrating a stable and suitable platform for the repeated determination of histamine. Additionally, the stability of the AN/AuPs/G biosensor was studied by measuring the current responses of the prepared biosensor for long-term storage. It retained 98.3% (Fig. 4D) of its initial response after 7 days of storage, indicating its good storage stability.

3.4 In vivo real-time monitoring at acupoint PC6 of rats

Dynamic changes in histamine at the acupoint would affect the acupoint-activation process for acupuncture effectiveness. Rats (Wistar SD rat from Beijing Vital River Laboratory Animal Technology Co., Ltd, Male, 6–7 weeks) were employed to monitor real-time dynamical changes in histamine at acupoints induced by acupuncture stimulation. As demonstrated in Fig. 5A and S4,† the AN or AN/AuPs/G electrodes were pierced 2–3 mm into the acupoint PC6, while the rat's tail was immersed in an electrolytic solution to establish a three-electrode system. PC6 was located on the inner part of the forelimb of the rat, between the ulna and radius suture, which was about 3 mm from the wrist. The AN or AN/AuPs/G electrodes were inserted perpendicular to the skin. All tests were conducted under the approval of the Animal Ethics Committee of Beijing University of Chinese Medicine (BUCM-2023120502-4174) and followed the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China.

Fig. 5 (A) The animal experiment diagram. (B) The differences of cyclic voltammograms between AN and AN/AuPs/G in vivo. The AN/AuPs/G was pierced for 2–3 mm from the PC6 acupoint. (C) The differences in the cyclic voltammograms before and after injecting histamine. (D) Real-time recording of AN/AuPs/G in vivo. Anesthetized rats were monitored for the changes in histamine concentrations at the PC6 acupoint. Inset: The relationship between time and oxidation peak value.

To investigate the potential of the AN/AuPs/G biosensors for real-time monitoring of histamine concentrations during acupuncture stimulation, we first anesthetized the rats with 2% pentobarbital. The AN/AuPs/G biosensors exhibited significantly higher CV responses, compared to the bare AN biosensors (Fig. 5B). The oxidation peak values of the AN/AuPs/G biosensors increased in a short period, indicating the release of histamine, along with acupuncture, which was consistent with previous literature.⁴³ Conversely, the oxidation peak values of the AN biosensors were almost unchanged, indicating their weak sensing properties. These results show the potential of the AN/AuPs/G biosensors for in vivo real-time monitoring of histamine.

To confirm that histamine, rather than other substances, was selectively detected at the acupoint, we plotted CVs before and after injecting 0.3 mL of histamine solution (100 mg L⁻¹), which is adjacent to acupoint PC6. After histamine injection, the oxidation peak value increased by 41% within 150 s (Fig. 5C), confirming the precise identification of histamine by the AN/AuPs/G biosensors. Additionally, the oxidation peak position shifted slightly to the right compared to in vitro detection, which is likely due to differences in pH (in vivo: 7.35–7.45, in vitro: 6.0) and the concentrations of histamine; this is a common phenomenon in animal experiments.

Subsequently, real-time monitoring of histamine concentrations was conducted by the AN/AuPs/G biosensor at non-acupoint and acupoint PC6 throughout the acupuncture stimulations, lasting 50 min each. The histamine concentration at acupoint PC6 (Fig. 5D) initially increased and then decreased during acupuncture, peaking at around 18 min, while minimal changes were observed at the non-acupoint (Fig. S5†). This specificity in histamine release between non-acupoint and acupoint PC6, as captured by the AN/AuPs/G biosensor, underscores its capability for real-time monitoring during acupuncture therapy. The rise in histamine concentration might be attributed to mast cell degranulation induced by acupuncture, while its subsequent decrease could be due to consumption when histamine played its role in relaxing blood vessels, enhancing vascular permeability, and affecting the nerve-endocrine-immunity regulation network. Therefore, the histamine concentration curves reflected its participation process in the acupoint initiation. The observed peak at around 18 minutes warrants further investigation, as it might inform optimal needle retention times for maximal therapeutic effect. Additionally, OCPT was employed to monitor histamine concentration in vivo (Fig. S6†), yielding results consistent with CVs. However, the AN/AuPs/G biosensors faced certain limitations. For instance, although the repeatability of the AN/AuPs/G biosensors was satisfactory in vitro, their repeatable performance in vivo must be optimized. Additionally, the detection limit and sensitivity of the AN/AuPs/G biosensors in vivo could be further improved by optimizing their construction. Further research to address these limitations is warranted in future studies.

4. Conclusions and future perspectives

Utilizing acupuncture needles as a foundation, we successfully developed highly sensitive AN/AuPs/G biosensors for histamine detection, achieving a competitive detection limit of approximately 4.352(±3.419) × 10⁻¹² mol L⁻¹ and good sensitivity of approximately 6.296 (±3.873) μA μM⁻¹, which compares favorably with other reported sensors in the literature. Furthermore, our biosensors exhibited excellent specificity, repeatability, and stability. Leveraging these attributes, we conducted a comparative analysis of real-time histamine concentration changes at acupoint PC6 and non-acupoint. Interestingly, minimal fluctuations were observed at the non-acupoint, whereas a distinct trend of initial increase, followed by a decrease was observed at acupoint PC6. This finding underscored the specificity of histamine release between non-acupoint and acupoint PC6. Importantly, the observed changes in histamine concentration at acupoint PC6 elucidated its involvement in the initiation of acupoint-related processes. Notably, the peak position, occurring approximately 18 minutes post-stimulation, held significant implications for optimizing needle retention time to achieve the best therapeutic outcomes. This study pioneered the real-time monitoring of histamine at acupoints with high sensitivity and offered invaluable insights into the acupoint-activation mechanisms underlying acupuncture efficacy.

Author contributions

P. L.: conceptualization; data curation; formal analysis; investigation; methodology; resources; software; validation; visualization; writing – original draft; writing – review & editing. A. Y.: data curation; investigation; resources. L. H.: formal analysis; software. B. Zhao.: software. Q. W.: software. Q. F.: investigation. S. N.: investigation. G. Y.: investigation. R. Z.: investigation. L. Y.: funding acquisition; resources; writing – review & editing. A. C.: supervision; writing – review & editing. C. L.: writing – review & editing. W. X.: conceptualization; funding acquisition; methodology; project administration; supervision; validation; writing – review & editing. All authors have approved the final version of the manuscript.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors had no conflicts of interest relevant to this article.

Acknowledgements

This work was supported by High-level Talent Project of Beijing University of Chinese Medicine (2022-XJ-KYQD-004), “Unveiling the List and Hanging the Leader's Flag” Project of Beijing University of Chinese Medicine (2024-JYB-JBZD-066), High-level Key Discipline Program of Acupuncture and Moxibustion from the State Administration of Traditional Chinese Medicine (zyyzdxk-2023254), National Natural Science Foundation of China (grant no. 52202043), and R&D Program of Beijing Municipal Education Commission (grant no. KM202310028012).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nr01998a

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