Introduction

The continuous increase in cancer incidence and mortality rates have become one of the major challenges to global human survival in the 21st century [1,2,3]. Early diagnosis of cancer not only helps reduce the mortality rate of cancer patients but also aids in lowering the treatment costs for patients [4, 5]. Therefore, on the one hand, a large amount of research is focused on exploring new biomarkers for early cancer diagnosis [6, 7]. Heat shock protein 70 (HSP70), a highly conserved protein and a biomarker for cancer, is found to be overexpressed in almost all types of tumors, such as prostate cancer, breast cancer, colorectal cancer, melanoma, bladder cancer, and head and neck squamous cell carcinoma [8,9,10]. Furthermore, the levels of HSP70 decrease significantly when tumor tissue is removed through surgery or chemotherapy [11]. This variation in HSP70 levels indicates that HSP70 protein can serve as a marker for assessing the response to cancer treatment [12, 13]. Therefore, the detection of HSP70 for early diagnosis and treatment response monitoring of all the aforementioned types of cancer is highly advantageous [14, 15]. However, traditional detection techniques for HSP70, such as ELISA, protein imprinting, protein microarrays, etc., have limitations such as low sensitivity, cumbersome procedures, large sample requirements, and high costs [16,17,18]. Therefore, the research and development of a rapid, highly sensitive, and cost-effective HSP70 sensor has become an urgent priority [3, 19]. Sun et al. [19] developed a novel electrochemical immunosensor for early screening of HSP70 using polyaniline-functionalized graphene quantum dots (PAGD) synthesized via in-situ polymerization. They modified the PAGD onto a graphite electrode and conducted HSP70 detection through an electrochemical system connected to an external reference electrode and a counter electrode. The sensor exhibited wide linear detection ranging from 0.0976 to 100 ng mL− 1 with a detection limit of 0.05 ng mL− 1. Sun et al. [20] investigated a novel electrochemical immunosensor for early screening of HSP70 based on porous graphene (PG) with a large specific surface area and excellent structure. Utilizing the strong adsorption and good bioreactivity of PG initially prepared via a simple thermal decomposition process, multiple heat shock protein 70 (HSP70) molecules could be firmly loaded onto PG to construct an alkaline electrode (HSP70/PG/GCE). Similarly, Karaboğa et al. [21] employed disposable indium tin oxide (ITO) coated polyethylene terephthalate (PET) electrodes modified with gold nanoparticles to construct a biosensor for HSP70 analysis. Özcan et al. [22] utilized a glassy carbon electrode as the working electrode, which was coated with graphene oxide. They employed EDC/NHS chemistry to covalently immobilize AntiHSP70 as the biorecognition element of the biosensor onto the graphene oxide layer. Subsequently, HSP70 detection was carried out through an electrochemical system connected to an external reference electrode and a counter electrode. These sensors, however, are not conducive to bedside testing as the working, reference, and counter electrodes are not integrated onto a single chip. Bedside testing sensors require integration of all components onto a small chip to enable rapid, portable, and real-time diagnostics. Sensors integrating a working electrode, a reference electrode and a counter electrode on a chip for the immediate detection of ovarian cancer markers or the liver cancer marker AFP have been reported recently [23, 24], however, the integration of three electrodes on a single chip for the detection of HSP70 has not yet been reported. Therefore, the development of integrated HSP70 sensors suitable for bedside care still requires further research.

It is well known that the concentration of the HSP70 biomarker is typically below 5 ng mL− 1. For detecting analytes at such low concentrations, electrochemical sensors often require signal amplification strategies. Currently, common strategies and methods for effectively enhancing signal transduction involve the addition of metal nanoparticles [14, 25] and polystyrene (PS) [26, 27] nanoparticles, among other nanomaterials, to increase the conductivity of the matrix. Enhancement of the conductivity of PS is achieved by incorporating conductive materials such as metal nanoparticles, conductive polymers, or carbon nanotubes into its structure [28]. Another method to increase detection signals in electrochemical biosensors relying on antigen-antibody specific reactions is to increase antibody loading on the electrode surface to generate a more functionalized surface. Functionalization of microspheres’ surfaces with biomolecular recognition elements such as antibodies enables selective capture of specific molecules or ions [29]. When these target molecules interact with the sensor surface, they can cause changes in current, voltage, or resistance, thereby allowing for the detection and quantitative analysis of target molecules at low concentrations [30, 31]. Nevertheless, the utilization of PS (a low-cost material) as a signal amplification material for HSP70 electrochemical biosensor research has not been reported yet.

In this study, a biosensor chip integrating working, reference, and auxiliary electrodes for HSP70 detection is developed. The working electrode on the chip was electrochemically modified using PS-AuNPs@Cys material, followed by incubation with HSP70 antibodies, forming an HSP70 antibody/PS-AuNPs@Cys/Au composite membrane on the working electrode of ITO. Differential pulse voltammetry (DPV) was employed to quantify the interaction between the sensor and different concentrations of recombinant HSP70 antigen, achieving highly sensitive detection of HSP70. The modification process of the working electrode on the biosensor chip and DPV detection of HSP70 are illustrated in Scheme 1.

Scheme 1
scheme 1

The schematic diagram of the manufacturing procedure and testing process for the HSP70 sensor

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Experimental

Reagents and materials

Amine group functionalized polystyrene nanoparticles (PS, 100 nm in size; solid content 50 mg mL− 1) were purchased from ZhongKeLeiMing Technology Co. (Beijing, China). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4⋅3H2O) and L-Cysteine were bought from Shanghai Jiachen Chemical Co. (Shanghai, China). Heat shock protein 70 (HSP70) (Ag), biotinylated HSP70 monoclonal antibody (Biotin-Ab), horse radish peroxidase labeled streptavidin (HRP–Strept), heat shock protein 70 (HSP70) enzyme-linked immunosorbent assay (ELISA) kits and bovine serum albumin (BSA) were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). 1-Ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), L-cysteine and glutaraldehyde (GA) were purchased from Sigma-Aldrich (U.S.A). Phosphate buffered saline (PBS buffer, pH 7.4) was prepared in deionized water produced by a Millipore Milli-Q ion exchange apparatus.

All reagents were of analytical reagent grade. The human venous blood samples were kindly provided by Sichuan Provincial People’s Hospital (Chengdu, China).

Apparatus

All electrochemical experiments were performed with electrochemistry workstation (Shanghai, CHI660D), using ITO three-electrode chip electrochemical sensor. The nanomaterials were characterized with a scanning electron microscope (SEM, HITACHI SU8010, Japan). The UV–vis absorbance spectra were recorded with ultraviolet-visible spectrophotometer (SPECORD 250 PLUS, German). Fourier transform infrared spectra (FT-IR) were recorded with a PerkinElmer FT-IR spectrophotometer (Nicolet is5, USA). The HSP70 ELISA reaction was recorded at 450 nm using microplate reader (Multiskan SkyHigh, USA).

Design and fabrication of the ITO three-electrode chip electrochemical sensor

The design of the bare three-electrodes ITO chip, shape and dimensions (shown in Fig. S1), was fabricated and produced by Huanan Xiangcheng Technology Co. Ltd., CA (Changsha, China). The preparation of the Ag/AgCl reference electrode was conducted as follows (illustrated in Fig. S1): First, a hole with the same size and shape of the reference electrode was made on a piece of tape using a puncher. Next, the tape with the hole was positioned over the bare ITO reference site and pressed down. Then, Ag/AgCl paste (SINWEI-3701) was added to the hole in the tape, making contact with the ITO. A spatula was used to spread the paste evenly into the hole. Afterwards, the tape was carefully removed and the Ag/AgCl was dried in an oven at 150 ℃ for 30 min. To verify the performance of the reference electrode, it was subjected to five cycles of cyclic voltammetry (with a potential range of -0.4 V to 0.4 V and a scan rate of 100 mV/s) in a solution of 5 mM [Fe(CN)6]3−/ [Fe(CN)6]4− and 0.1 M KCl.

Synthesis of AuNPs

The procedure involves subjecting a 50 mL volume of a 0.02% aqueous solution of HAuCl4·3H2O to thermal treatment, reaching a temperature of 100 °C. Subsequently, 2 mL of a 1% sodium citrate solution is introduced into the heated solution with continuous stirring for a duration of 15 min. Following this, the heating is discontinued, and stirring is maintained for an additional 30 min. The resultant AuNPs are then stored at 4 °C. This method entails the utilization of sodium citrate as a reducing agent, furnishing electrons to facilitate the reduction of Au(III) ions to Au(0) atoms to yield nanoscale gold nanoparticles. Furthermore, sodium citrate fulfills the role of a dispersing agent, thereby aiding in the prevention of coagulation or aggregation of the newly formed gold nanoparticles during the synthesis process. This mechanism effectively ensures that the resultant nanoscale gold particles maintain a state of dispersion, thereby precluding the formation of clusters. The manipulation of the concentration of sodium citrate and the adjustment of reaction conditions was employed to exercise control over both the size and morphology of the produced AuNPs. By fine tuning the concentration of sodium citrate and modulating the reaction parameters, precision in the control of particle size was attained.

Synthesis AuNPs@Cys and PS-AuNPs@Cys

To functionalize nanoscale gold particles, 50 mM cysteine was added to the pre-prepared nanoscale gold solution in a 1:1 ratio followed by stirring for 2 h. This procedure leverages covalent bonds referred to as thiol-gold bonds, formed through the interaction of sulfur atoms with gold atoms. The remarkable affinity of sulfur atoms for gold atoms results in the establishment of robust and enduring covalent bonds. This inherent strength and stability of the thiol-gold bond render it highly advantageous in various applications involving nanoscale particle synthesis and functionalization. Such applications encompass, but are not limited to, surface modification of nanoscale gold particles, the development of biosensors, drug delivery systems, and a spectrum of other specialized functions.

The cysteine-functionalized nanoscale gold was functionalized with polystyrene. 400 µL of amine group functionalized polystyrene, accompanied by 100 µL of cross-linking agents, EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide), and NHS (N-Hydroxysuccinimide), were added to a 10 mL solution of cysteine-modified nanoscale gold. The resulting mixture was subjected to stirring for 30 min. EDC and NHS were used as cross-linking agents to stabilize and augment the specificity of the amide bonds.

Electrochemical deposition of PS-AuNPs@Cys on ITO chip

The three-electrode ITO chip is first immersed in an isopropanol solution and sonicated for one hour. The electrode is then immersed in anhydrous ethanol and sonicated for another hour. This process is followed by an additional hour of sonication in ultrapure water. Subsequently, the ITO chip is dried at 100 °C, making it ready for subsequent use. For the electrochemical deposition of PS-AuNPs@Cys on the working electrode of ITO chip, a potential of -1.4 V is chosen, and a deposition duration of 120 s is used for constant potential deposition to obtain a PS-AuNPs@Cys/Au film modified working electrode.

Immobilization of AntiHSP70 proteins on PS-AuNPs@Cys/Au modified ITO electrode

After the modification of the working electrode of the ITO chip by PS-AuNPs@Cys, a precise sequence for the immobilization of AntiHSP70 proteins is implemented as follows. Initially, 10 µL of a 2.5% glutaraldehyde solution is introduced, and a cross-linking period of 30 min is allowed. Subsequently, the unbound glutaraldehyde is removed through a rinsing process employing PBS. Then, 10 µL of a 20 µg mL− 1 solution of AntiHSP70 was added, followed by an incubation at 4 °C for a duration of 12 h. To further ensure specificity and block any non-specific interactions, a 1% BSA solution was introduced to prevent other thiol-containing substances from binding to the PS-AuNPs@Cys/Au/ITO. After this blocking step, any unbound BSA is meticulously removed through a series of washing with PBS. Finally, 10 µL of HSP70 solution is added to the PS-AuNPs@Cys/Au/ITO surface previously incubated with the antibody.

DPV measurement

The prepared electrode was exposed to varying concentrations of HSP70 solutions to assess the HSP70 levels. Specifically, a series of 10 µL HSP70 solutions with different concentrations were deposited, followed by a 1-hour incubation at 37 °C. Subsequently, the electrode was rinsed with a 0.01 M PBS buffer to eliminate any unbound antigens. The potential is then scanned within the range of 0.2 to 0.6 V. The parameters for DPV were set as follows: an amplitude of 50 mV, a pulse width of 50 ms, a scan width of 16.7 s, and a pulse period of 500 ms. DPV signals corresponding to the concentration of HSP70 were recorded and quantified.

Preparation of the samples

The serum samples were prepared following the commonly used method in the hospital. Venous blood was collected in anticoagulant tubes as samples, and within 30 min of collection, it was centrifuged at 1000×g, 2–8℃ for 15 min. The supernatant was taken as the serum sample for testing. The serum samples were subjected to electrochemical testing following the method described in Sect. DPV measurement and to ELISA testing using the methods provided in the ELISA kit. The results of both tests were analyzed and compared.

Results and discussion

UV and FTIR characterization of AuNPs@Cys and PS-AuNPs@Cys

Characterization of AuNPs@Cys and PS-AuNPs@Cys nanoparticles performed by the UV–vis spectroscopy and FTIR technique are shown in Fig. 1. The absorption spectrum of AuNPs@Cys colloidal solutions shows the same characteristic plasmon resonance band centered at 525 nm as AuNPs, but shown wine red for AuNPs and blue purple colour AuNPs@Cys respectively. The UV spectrum of PS-AuNPs@Cys solutions shows a characteristic plasmon resonance band centered at 525 nm and 680 nm with a purplish red colour (Fig. 1A). The new peak at 680 nm was resulted from the coupling of PS microspheres with AuNPs@Cys through a crosslinking agent. This, signifies the successful preparation of PS-AuNPs@Cys. The FTIR spectra of PS-AuNPs@Cys is as shown in the Fig. 1B. The detailed FTIR attributes of the AuNPs@Cys and PS nanoparticles are presented in supplement material (see Fig. S2). The FTIR infrared spectra of PS-AuNPs@Cys is shown in Fig. 1B. Comparing the differences in infrared spectra between PS-AuNPs@Cys and AuNPs@Cys (Fig. S2A) and PS (Fig. S2B), it was observed that the peak intensity at 3422 cm− 1 weakened, while new absorption peaks appeared at 3000 cm− 1, 1711 cm− 1 and 1609 cm− 1. The peak at 3000 cm− 1 is attributed to the stretching vibration of C-H bonds in the benzene ring, while the peak at 3422 cm− 1 corresponds to the stretching and bending vibrations of N-H bonds, the peaks at 1711 cm− 1 and 1609 cm− 1 belong to the vibrations of amide bonds. Consequently, the weakened peak intensity at 3422 cm− 1 and the appearance of new absorption peaks at 1711 cm− 1 and 1609 cm− 1 indicate the binding of amine-functionalized PS to AuNPs@Cys through amide bonds.

Fig. 1
figure 1

(A) The UV–vis absorption spectra of AuNPs. (B) FT-IR spectra of PS-AuNPs@Cys

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The mechanism of PS/AuNPs @Cys/Au film formation on ITO

When the ITO electrode is immersed in a dispersion of PS-AuNPs@Cys and a constant potential of -1.2 V to -1.4 V is applied, a PS-AuNPs@Cys/Au film can be formed on the ITO electrode. The electrochemical formation mechanism is as follows: Initially, the amine-functionalized polystyrene (PS) molecules in the solution are adsorbed onto the electrode surface due to their affinity for the electrode material. This adsorption creates a positively charged layer on the electrode. Subsequently, the negatively charged AuNPs@Cys, which are gold nanoparticles modified with cysteine, in the solution are attracted to the positively charged PS layer on the electrode surface through electrostatic interactions. The cysteine-modified gold nanoparticles adsorbed on the PS layer are then reduced to gold at a constant potential of -1.4 V (Ag/AgCl), resulting in the deposition of gold on the ITO surface. This process leads to the formation of a PS-AuNPs@Cys/Au/ITO electrode. Adsorption: The presence of PS-AuNPs@Cys molecules in the solution adsorb onto the electrode surface, forming a positively charged layer on the electrode.

Reduction of AuNPs@Cys: AuNPs@Cys + e− → Au + Cys−.

Deposition of Au on ITO: Au+ + e− → Au.

This potential-controlled deposition method provides a simple approach for fabricating functional films with modifications on the patterned electrode surface.

Morphological and electrochemical characterization the current amplification effect of PS-AuNPs@Cys/Au film on ITO electrode

The morphologies of AuNPs@Cys, PS, and PS-AuNPs@Cys/Au were studied using scanning electron microscopy (SEM). As depicted in Fig. 2A, AuNPs@Cys nanoparticles were uniformly deposited on ITO substrate. The PS nanoparticles exhibited a nanospherical structure with an average diameter of 100 nm. With respect to the AuNPs@Cys and PS, in PS-AuNPs@Cys nanoparticles, AuNPs@Cys clustering around PS forms a more compact structure, making it easier for electrons transfer in PS-AuNPs@Cys/Au membrane, thereby reducing its resistance and producing an amplified current response, thereby reducing its resistance and producing an amplified current response. These are consistent with the EIS and CV testing results as shown in Fig. 2D and E, respectively.

Fig. 2
figure 2

The SEM images of (A) AuNPs@Cys, (B) PS and (C) PS-AuNPs@Cys/Au, (D)EIS of AuNPs@Cys, ITO and PS-AuNPs@Cys/Au, (E) CV of AuNPs@Cys, ITO and PS-AuNPs@Cys/Au

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Optimization of the PS-AuNPs@Cys/Au/ITO immunosensors

The uniform incubation of HSP70 antibody at the optimal concentration on PS-AuNPs@L-Cys/Au modified ITO electrode, as well as the blocking of non-active sites with BSA are two crucial steps for optimizing the immunosensor. Accordingly, the concentrations of the antibody and blocking agent, as well as the antibody incubation time are acrucial. Various concentrations of HSP70 antibodies (5 µg mL− 1, 10 µg mL− 1, 15 µg mL− 1, 20 µg mL− 1, 25 µg mL− 1) were prepared in phosphate-buffered saline (PBS), and incubated onto the PS-AuNPs@L-Cys/Au modified ITO surfaces. The study of incubation time involves incubating different concentrations of antibodies at different reaction times (i.e. 3, 6, 9, 12, and 15 h). The influence of antibody concentration and reaction time detected by electrochemical immunosensor DPV response. The change in current was calculated using the equation ΔI = I0 - Ii, where I0 and Ii represent the peak current magnitudes before and after the interaction with HSP70 antibodies respectively. The ΔI values increased with longer incubation times and reached a plateau at 9 h (Fig. 3), indicating full antibody coverage on the PS-AuNPs@Cys/Au modified ITO surface. Considering the convenience of experimental scheduling, we have chosen to set the incubation time to 12 h. Furthermore, the optimal antibody concentration for incubation on the electrochemical sensor surface was investigated. The ΔI values gradually increased with rising antibody concentration, reaching saturation at 20 µg mL− 1. Based on these results, 20 µg mL− 1 was chosen as the optimal concentration for modifying the antibodies. To minimize nonspecific binding, 1% BSA was used to block free aldehyde groups, and the incubation time was optimized to 30 min.

Fig. 3
figure 3

The ΔI response of antibody concentration and reaction time using differential pulse voltammetry (DPV)

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DPV response of PS-AuNPs@Cys/Au/ITO immunosensors on HSP70

Figure 4 illustrates the interaction between HSP70 antibody/PS-AuNPs @Cys/Au-modified ITO electrode and different concentrations of HSP70 antigen in solution. Peak current was used to assess the interaction between the antibody and antigen. The peak current of DPV without HSP70 in potassium ferricyanide solution was the highest, and it decreased with increasing HSP70 antigen concentration (Fig. 4A). This decrease was attributed to the binding of more antigen molecules to the immobilized antibody at higher concentrations of HSP70 antigen. The relative current was plotted against the antigen concentration to obtain a standard curve (Fig. 4B). The linear range was observed between 0.1 and 1000 ng mL− 1. The linear relationship is described by the equation Ip = -4.652×log (CHSP70) + 40.824 (where Ip = peak current, and CHSP70 = HSP70 concentration in ng mL− 1). This linear equation exhibits a high correlation coefficient (R2) of 0.996. A control test was conducted on the HSP70 antibody/Au/ITO immunosensor without PS modification (Fig. 4C). No linear correlation was observed, and a higher error bar was mainly due to the inability of AuNPs@Cys to electrodeposit Au on ITO electrode because of its negative charge.

The calculation of the limit of detection (LOD) was calculated using the formula, LOD = (κ * σ) / S, where S = the sensitivity, which corresponds to the slope of the current versus antibody curve. The standard deviation of blank measurements is denoted as σ, and the confidence level parameter κ is set to 3 for a statistical confidence level of 99.6%. The calculated LOD was 25.7 pg mL− 1. For the Limit of Quantification (LOQ), HSP70 samples at concentrations of 0.01 ng mL− 1, 0.05 ng mL− 1, and 0.1 ng mL− 1, respectively were tested. While there was a noticeable current response for the 0.05 ng mL− 1 and 0.01 ng mL− 1 HSP70 samples, the results of repeated experiments showed obviously fluctuations, indicating the inability to accurately detect such low concentrations of HSP70. The 0.1 ng mL− 1 HSP70 sample could be accurately detected. Hence, the LOQ of this sensor was considered to be 0.1 ng mL− 1.

In comparison to other reported methods, this approach showcases a broader linear range and a relatively low detection limit. Moreover, simple modification of the working electrode and reference electrode of the three-electrode chip composed of indium tin oxide (ITO) enables point-of-care testing in clinical practice, thereby expanding the sensor’s applicability. In this study, modification of the working electrode with PS-AuNPs@Cys/Au increased the number of antibody loads, resulting in higher sensitivity.

Fig. 4
figure 4

(A) DPV responses of the HSP70 antibody/PS-AuNPs @Cys/Au/ITO immunosensor at different concentrations, ranging from a to h: 0ng mL− 1, 0.1 ng mL− 1, 0.5 ng mL− 1, 1 ng mL− 1, 5 ng mL− 1, 10 ng mL− 1, 100 ng mL− 1, and 1000 ng mL− 1. (B) Linear plot of DPV current against the logarithm of HSP70 concentration (R2 = 0.996). (C) DPV responses of the HSP70 antibody/Au/ITO immunosensor at different concentrations, ranging from a to h: 0ng mL− 1, 0.1 ng mL− 1, 0.5 ng mL− 1, 1 ng mL− 1, 5 ng mL− 1, 10 ng mL− 1, 100 ng mL− 1, and 1000 ng mL− 1. (D) Plot of the response of different concentrations of HSP70 at the AuNPs@Cys immunosensor

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Selectivity, stability and reproducibility of the PS-AuNRs@Cys/Au immunosensor

We investigated the potential interference of other members of the HSP family (such as HSP90) as well as metabolites in serum including IL-2, TNF-α, glucose, and urea on the HSP70 differential pulse voltammetry (DPV) signal to validate the selectivity of the sensor. Based on the reference concentration ranges of these metabolites in normal human serum, we chose interference concentrations of 0.5 ng mL− 1 IL-2, 0.5 ng mL− 1 TNF-α, 10 mg mL− 1 glucose, 2 mg mL− 1 urea, and 16 ng mL− 1 HSP90. After incubating 10 µL of the above solutions on different immunosensor chips, we detected a slight change in current (ΔI). However, when these metabolites were mixed with 10 ng mL− 1 HSP70 for detection, a significant increase in current (ΔI) was observed, as shown in Fig. 5. This indicates excellent selectivity of the sensor. We also performed repetitive DPV measurements of 1 ng mL− 1 HSP70 every two days over a two-week period using the PS-AuNRs@Cys/Au-modified immunosensor. The measured current changes showed a relative standard deviation (RSD) of 6.74%, demonstrating the excellent stability of the prepared sensor.

Fig. 5
figure 5

Selectivity of the immunosensor for HSP70 detection

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To evaluate the reproducibility of the sensor, six sets of sensors using identical experimental conditions were prepared. 1ng mL− 1 of HSP70 standard was introduced into simulated serum-like biological samples for DPV detection The result is as shown in Table 1. The assessment of HSP70 recovery in the serum samples revealed a range of recoveries from 96.33 to 113.42%. This indicates that the immunosensor offers acceptable accuracy for the clinical quantification of HSP70, underlining its reliability and consistency.

Table 1 Determination of HSP70 in simulated serum samples
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Analysis of real samples

Importantly, to further assess the practicality of the newly developed immunosensor for clinical applications, nine different human serum samples were analyzed for detection of HSP70 using the developed immunoassay and the enzyme-linked immunosorbent assay (ELISA) method. The comparison of using the proposed immunosensor and the reference method (ELISA) to detect HSP70 is presented in Table S1 and Figure S3. For individuals with low HSP70 levels, ELISA method failed to measure, whereas our newly developed immunosensor could provide detection data, indicating the superiority of our developed immunosensor over ELISA. Additionally, the present immunosensor shows advantages in terms of linear detection range, detection limit, recovery rate in comparison to the literature [19,32], as shown in Table 2. This further confirms the superiority of this sensor.

Table 2 Analytical performances of different methods for the detection of HSP70
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Conclusions

In this work, we developed an on-chip electrochemical sensor as a highly sensitive platform for detecting HSP70. A self-synthesized composite nanomaterial, PS-AuNPs@Cys, was deposited onto the working electrode of an ITO chip using constant potential method. This not only enhanced the conductivity of the electrode but also served as a carrier material to enhance the loading of HSP70 antibodies, resulting in signal amplification. The ITO chip for detecting HSP70 is easy to prepare, cost-effective, and exhibits detection limits superior to those reported in the literature [19]. Its low sensitivity and the distinguishable test results in different patient serum samples demonstrate potential application value of this biosensor for bedside HSP70 detection.