Polyadenine-mediated aptamer-conjugated gold nanoparticles for the detection of interleukin-6

Abstract

Acting as a pivotal cytokine of innate immunity, the signal transduction of interleukin 6 (IL-6) is always elevated in relation to the occurrence of injury, bacteria and chemical invasion. IL-6 as a critical care biomarker can be used for the clinical diagnosis of diseases ranging from bacterial infections to most inflammatory reactions. Then, a simple, cost-effective and sensitive colorimetric aptasensor was designed for the specific detection of IL-6. The IL-6 aptamer anchored on gold nanoparticles (AuNPs) by polyadenine accurately recognized IL-6, and then, the folded IL-6 aptamer enhanced the stability of AuNPs against salt-induced aggregation. The degree of AuNP aggregation increased inversely with the concentration of IL-6. A higher IL-6 content resulted in lower aggregation of AuNPs, which can be measured by spectrophotometry. The results showed that the proposed aptasensor exhibited a linear detectable range of 7–500 pg mL⁻¹ with a limit of detection of 4.652 pg m⁻¹. Furthermore, this proposed colorimetric aptasensor achieved the discrimination of IL-6 from other analogues and could detect IL-6 in human serum samples, which demonstrated its potential application for the detection of IL-6 in complex matrices.

---

1. Introduction

The adverse effects of chronic inflammation are the driving forces for the initiation and progression of several chronic diseases, such as cardiovascular disease, kidney disease, diabetes and cancer,^1,2 as well as acute mesenteric ischaemia³ and traumatic brain injury.⁴ In the past two decades, there has been increasing recognition that interleukin (IL)-6, discovered as a product of T cells that uniquely induces the differentiation of B lymphocytes and enhances active antibody production,^5,6 is involved in the genesis of these interrelated disorders. IL-6 has emerged as the primary regulator of inflammation and is one of the few truly pleiomorphic cytokines that mediate a wide range of biological activities. Several epidemiological studies have indicated that an elevation in serum IL-6 concentration is associated with an increase in the incidence of sepsis, coronary heart disease, and metastatic melanoma.^1,7,8 The serum concentrations of IL-6 in normal healthy individuals are typically quite low, around 0.2–7.8 pg mL⁻¹.^6,9 However, IL-6 concentrations in adults with sepsis and metastatic melanoma can exceed 1600 pg mL⁻¹.^7,8 Moreover, the serum concentration of IL-6 in patients diagnosed with acute mesenteric ischaemia at the transmural stage could even approach 20 000 pg mL⁻¹.³ Determination of IL-6 levels could be useful in the diagnosis of these kinds of infections and diseases and could improve the outcomes of diagnosis and the management efficiency of patients with systemic inflammatory response syndrome at different stages.^3,7 Therefore, fast, sensitive and accurate detection of IL-6 across a broad dynamic concentration range is highly desirable for the early diagnosis of the progression of these diseases involved with IL-6 and assess the specific treatments.^10,11

Enzyme-linked immunosorbent assay (ELISA) is considered the most conventional and standard assay for the detection of IL-6, with a limit of detection of about less than 10 pg mL⁻¹.^9,12 However, it has limitations in terms of hour-long procedures and high costs. For example, the assay duration of the Elabscience IL-6 Human ELISA kit (Elabscience Biotechnology Co., Ltd, China) is approximately 3.5 hours, and its price is RMB 2400.00 for one pack. Several biosensors have been developed in recent years for the detection of IL-6 as alternative approaches, such as surface-enhanced Raman spectroscopy (SERS),^13–15 electrochemical impedance spectroscopy,^11,16,17 molecularly imprinted polymer-based sensors¹⁸ and fluorescence resonance energy transfer-based sensors.¹⁹ Although these assays are highly sensitive, they generally have the disadvantages of cumbersome preparation and operation and require specialized and sophisticated instruments.^20,21 Therefore, there is a strong drive to develop a cost-effective, convenient and reliable technology to detect IL-6 in complex biological systems.

Gold nanoparticle (AuNP)-based colorimetric methods have been extensively developed for biomolecule identification due to their simplicity, adaptability, low cost and versatility.^22,23 The colorimetric assay based on AuNPs often relies on the analytes as the cross-linking molecules with multiple binding sites to induce the aggregation of AuNPs.^24–27 IL-6 has a low molecular weight of 20 kDa⁶ and cannot provide sufficient recognition sites as “crosslinking molecules” to induce the aggregation of AuNPs. To overcome the insufficient bridges used as cross-linking agents to aggregate AuNPs, Alejandra Alba-Patiño et al. proposed a sandwich immunoassay for the detection of IL-6 using a smartphone as a portable reader to digitize the color change of plasmonic nanoparticles.¹⁰ Although antibodies remain the gold standard sensing units, there is growing interest in introducing aptamers as specific biomedical and molecular recognition elements due to their high affinity and specificity towards their ligands.²⁸ Susan Giorgi-Coll et al. immobilized two types of IL-6 aptamers on AuNPs via Au–S bond to design a “sandwich-style” optical assay. The binding of IL-6 to the complementary aptamer pair induced the aggregation of AuNPs and consequently a visible color change.²⁹ Although this method avoids the restriction of multiple binding sites between aptamers and their targets, it suffers from a deficiency in sensitivity because its detection limit is 1950 pg mL⁻¹. This low sensitivity may be ascribed to fewer epitopes being exposed to IL-6 to prevent it from sufficiently binding to these two probes of AuNPs simultaneously. Although a split-aptamer-based strategy has been developed to address this issue, this elegant approach is suitable only for a few molecules, except IL-6.³⁰ Marjan Malekmohamadi et al. designed a salt-induced non-crosslinking AuNP aggregation colorimetric assay for IL-6. The aptamer immobilized on AuNPs through a coordination bond was desorbed from the AuNP surface in the presence of IL-6; thus, AuNPs underwent aggregation at a certain amount of salt concentration due to the diminishment of the electrostatic repulsion force.³¹ The limit of detection of this assay can reach 70 pg mL⁻¹, while it may be susceptible to the overdose of AuNPs and aptamers, yielding a reduction in detection capability.

Traditionally, the conjugation of DNA with AuNPs occurs through the well-established strong Au–S chemistry. The thiolated oligonucleotides self-assemble at the surface of AuNPs via a salt aging process.³² Adenine, as one kind of DNA base, has an exceptionally high adsorption affinity towards gold due to its exocyclic amino group located at the 6th position and a ring nitrogen in the 7th position.³³ The specific adsorption of polyadenine sequences (polyA) on the AuNP surface enables it to serve as a linker between AuNPs and DNA, and the appended recognition block forms an upright conformation that favors target recognition.³⁴ Our group previously reported competitive non-cross-linking deaggregating sensing strategies for polyadenine-mediated AuNP-based assays by establishing competition between the targets and complementary ssDNA/precursor of the target.^35,36 A key feature associated with an aptamer is that its conformation undergoes a transition from random coil into compact tertiary structures upon recognizing its target,²⁸ which could be exploited to design a sensor by maintaining the distance between each AuNP in contrast to traditional cross-linking aggregation and competitive sensing strategies.

Aiming at improving the analytical performance of AuNP-based colorimetric biosensing strategies under the circumstance of limited binding sites between aptamers and targets of IL-6, specific aptamer–protein interactions were employed to stabilize aptamer-functionalized AuNPs in the presence of salt for the development of a label-free colorimetric assay. The polyA-mediated aptamer for IL-6 was anchored around AuNPs using a polyA sequence. The DNA-modified AuNPs exhibited a high tolerance to a high-salt solution (MgCl₂ concentrations up to 10 mM). This stability of DNA-modified AuNPs was diminished by the further addition of MgCl₂ as a trigger, resulting in the aggregation of DNA-modified AuNPs and a red-to-purple color change. In contrast, the polyA-mediated aptamer folded upon binding with IL-6. The steric hindrance provided by the folded aptamer made DNA-modified AuNPs more stable against salt-induced aggregation. Therefore, the color of the solution remained original. The affinity binding between the aptamer and IL-6, as well as the experimental conditions, was investigated using a colorimetric approach. The detection of IL-6 was achieved as low as 4.652 pg mL⁻¹ in the range of 7–500 pg mL⁻¹. This assay enables the detection of IL-6 spiked in serum samples, exhibits excellent analytical performance towards IL-6 determination and might become a potential alternative tool for the detection of IL-6 in biomedical research and early clinical diagnosis.

2. Materials and methods

2.1 Materials and reagents

Gold(III) chloride trihydrate (HAuCl₄·3H₂O), trisodium citrate anhydrous, and citric acid were purchased from Aladdin (Shanghai, China). MgCl₂ was purchased from Macklin (Shanghai, China). HCl and NaOH were purchased from Chongqing Chuandong Chemical (Group) Co., Ltd. Phosphate-buffered saline tablets and TE (pH 8.0) were purchased from Solarbio (Beijing, China). Recombinant human IL-4, IL-6, IL-8, IL-10, and the HPLC-purified interleukin 6 aptamer mediated by polyA (polyA-Apt) were purchased from Sangon Biotechnology Co., Ltd (Shanghai, China). The IL-6 ELISA kit (E-EL-H6156) was purchased from Wuhan Elabscience Biotechnology Co., Ltd (Wuhan, China). The detailed sequence of the polyA-Apt was 5′-AAA AAA AAA A TTT TT CTTCCAACGCTCGTATTGTCAGTCTTTAGT-3′.

All other reagents were of analytical reagent grade. All solutions were prepared using ultrapure water with resistivity of 18.2 MΩ cm.

2.2 Instruments

Absorbance measurement was performed on a custom-made absorption spectrometer with a 10 mm path length cuvette at room temperature, which was previously used several times.^35,37 In the spectroscopy system, a tungsten halogen lamp (HL2000, Choptics Co., Ltd, Shanghai, China) and a spectrometer (EQ2000, Choptics Co., Ltd., Shanghai, China) were used as the light source and detector, respectively. The absorbance of the sample was analyzed using programs written in C++ and Matlab. Transmission electron microscopy (TEM) was carried out using a T-20 transmission electron microscope (FEI, USA). The hydrodynamic diameter was determined using a Zetasizer Nano ZS (Malvern Panalytical, USA).

2.3 Preparation of AuNPs and the conjugation of aptamer-modified gold nanoparticles

The citrate-capped AuNPs of ∼12 nm in diameter were synthesized by applying the sodium citrate reduction method.³⁵ The 10 mL HAuCl₄ aqueous solution (1 mM) was brought to reflux and heated to boiling. Then, 15 mL of 1% trisodium citrate solution was quickly added to this boiling HAuCl₄ solution for 20 min under vigorous stirring. This mixture solution was cooled naturally to room temperature. Finally, the product was filtered using a 0.22 µm PES syringe filter and stored in a dark glass bottle at 4 °C. The concentration of AuNPs was spectrometrically estimated as 8 nM based on the Lambert–Beer law.

The aptamer-functionalized gold nanoparticles (polyA-Apt@AuNPs) were prepared using the salt-aging method according to a previous method.³⁵ The DNA oligonucleotides of polyA-Apt were dissolved in TE buffer (5 mM MgCl₂, pH 8.0) to prepare a 100 µM DNA stock solution. Then, 19.5 µL of polyA-Apt solution was added to 1125 µL as-prepared AuNP solutions (8 nM) to approach the molar ratio of polyA-Apt to AuNPs as 200 : 1 and gently stirred at 150 rpm for about 24 h in the dark at 25 °C to ensure sufficient reaction. After that, 12 µL of 500 mM Tris-acetate buffer (pH 8.2) and 114 µL of 1 M NaCl solution were successively added dropwise to the mixture and stored in the dark at 25 °C for an additional 48 h in order to maximize the amount of aptamer loading on the surface of AuNPs. Afterward, the mixed solution was washed three times in ultrapure water through centrifugation at 16 900g for 30 min to remove the unassembled aptamer. Finally, the polyA-Apt@AuNPs were resuspended in PBS solution (10 mM, pH 7.2) for further use.

2.4 Detection of IL-6 standard samples

The detailed procedure for the assay of IL-6 was performed as follows. First, recombinant human IL-6 powder was dissolved in PBS buffer (10 mM, pH 7.2) as a stock solution. Second, 50 µL of polyA-Apt@AuNPs and 30 µL of IL-6 solution at various concentrations were added to 220 µL of PBS buffer (10 mM, pH 6.5) and mixed via brief vortexing. Then, the above solution was incubated at 37 °C for 90 min. Afterwards, 33 µL of MgCl₂ solution (100 mM) used as trigger was added to the above mixture to make the concentration of MgCl₂ in the mixture approximately 10 mM. After 30 min of MgCl₂ solution addition, the absorption spectra and the corresponding absorbance ratio at 650 nm and 520 nm (A₆₅₀/A₅₂₀) of the mixture were recorded by applying a custom-made absorption spectrometer, respectively. The signal corresponding to each IL-6 concentration level was defined as the absorbance ratio A₆₅₀/A₅₂₀, where A₆₅₀ and A₅₂₀ are the absorption measured at wavelengths of 650 nm and 520 nm, respectively. The same procedure was carried out for specificity testing of three other proteins, including IL-4, IL-8 and IL-10.

2.5 Analysis of IL-6 in serum samples

Serum samples were obtained from the University–Town Hospital of Chongqing Medical University and the Second Affiliated Hospital of Chongqing Medical University. Solutions with different concentrations of IL-6 were prepared in a diluted human serum solution (1 : 10 dilution with 10 mM PBS buffer). The serum samples containing IL-6 were tested according to the detection process of the IL-6 standard sample.

3. Results and discussion

3.1 Characterization of AuNPs and polyA-Apt@AuNPs

The optical properties of the bare AuNPs and polyA-Apt@AuNPs are characterized by UV-Vis absorption spectroscopy. The localized surface plasmon resonance bands are characteristics of AuNPs and are highly sensitive to their size, shape, and the environment surrounding them. The absorbance spectra of AuNPs and polyA-Apt@AuNPs were measured, as shown in Fig. 1a. The AuNPs exhibited a characteristic resonance peak at ∼520 nm, and the binding of polyA-Apt to the surface of AuNPs red-shifted the absorption wavelength by approximately 5–6 nm following functionalization. Table S1 compares the resonance wavelengths of naked AuNPs and polyA-Apt-functionalized AuNPs prepared in five batches. Their resonance wavelengths were almost consistent, and coefficients of variation were in the range of 0.34–0.58%, indicating that the prepared AuNPs have good reproducibility. The TEM images of AuNPs and polyA-Apt@AuNPs shown in Fig. 1b and c demonstrate that both were distributed uniformly. The average diameter of AuNPs was 12.15 ± 0.32 nm with a spherical shape after analyzing more than 100 nanoparticles from several TEM images using ImageJ software (Fig. S1a). The morphology of polyA-Apt@AuNPs was similar to that of AuNPs, and their diameter was about 12.48 ± 0.62 nm without significant change compared to that of as-synthesized AuNPs (Fig. S1b). Dynamic light scattering (DLS) measurements were also performed, and its data proved that the hydrodynamic diameters increased from 12 nm to 24 nm after AuNPs were covered by a polyA-Apt probe (Fig. 1d).

Fig. 1 (a) The characteristic resonance wavelength of the AuNPs at ∼520 nm and the polyA-Apt@AuNPs at ∼526 nm. TEM image of the well-dispersed (b) AuNPs and (c) polyA-Apt@AuNPs. (d) DLS measurements for hydrodynamic diameters of the AuNPs and polyA-Apt@AuNPs.

3.2 Principle of the IL-6 sensing strategy

The proposed sensing method for the IL-6 assay is illustrated in Fig. 2. The polyA-Apt probe consists of two fragments: a poly A tail and a recognition block. The polyA tail covalently attaches onto AuNPs, and the recognition block is used to capture the target of IL-6 through DNA-protein binding. In the absence of IL-6, the electrosteric stabilization of polyA-Apt@AuNPs is reduced by mixing the assay buffer due to the addition of appropriate MgCl₂, which causes polyA-Apt@AuNPs to aggregate and is accompanied by a change in the color of the solution from red to blue. In contrast, in the presence of IL-6, the recognition block of Apt on the AuNPs could specifically bind to its target, leading to the formation of Apt/IL-6 complex. Although MgCl₂ can diminish the force of electrostatic repulsion that stabilizes polyA-Apt@AuNPs, the Apt/IL-6 complex provides steric repulsion to hinder the aggregation process. As a result, the steric repulsion produced by the formation of Apt/IL-6 complex overcomes the van der Waals attraction between each polyA-Apt@AuNP and maintains the interparticle distance. Thus, the color of the solution remains wine red.

Fig. 2 Construction of the colorimetric sensor based on the polyA-Apt@AuNPs for the detection of IL-6.

The primary important aspect of this proposed method is that the binding of IL-6 could enhance the steric repulsion forces to stabilize the polyA-Apt@AuNP colloids against van der Waals attraction. Absorption spectroscopy and scanning electron microscopy were performed to investigate the feasibility of the proposed sensing strategy. As shown in Fig. 3a, the absorption spectra of polyA-Apt@AuNPs exhibited one resonance around 526 nm, as expected, (curve 1) and a wine red color was observed (the inset image). When a certain amount of MgCl₂ was added into the solution of polyA-Apt@AuNPs, the polyA-Apt@AuNPs displayed a purple-gray color with the disappearance of the resonance peak at 526 nm and a rise of another peak at approximately 650 nm (curve 2), which implied an aggregation of polyA-Apt@AuNPs. In contrast, once a 250 pg per mL IL-6 solution was mixed with polyA-Apt@AuNPs and incubated, followed by the addition of MgCl₂. The aggregation degree of polyA-Apt@AuNPs was reduced, where the resonance wavelength at ∼526 nm was predominant (curve 3) and the color of the colloid solution was pink. This demonstrated that binding of IL-6 with polyA-Apt on the surface of AuNPs forms Apt/IL-6 complex, leading to resistance against aggregation. This transition is consistent with the nanoparticle distribution observed by TEM images (Fig. 3b–d). Fig. 3b and c show that the monodispersed polyA-Apt@AuNPs aggregated after the addition of MgCl₂. Fig. 3d shows that the distance between each polyA-Apt@AuNP was enlarged in the presence of both IL-6 and MgCl₂ in comparison to that of Fig. 3c, where only MgCl₂ was added.

Fig. 3 (a) Absorption spectra of the polyA-Apt@AuNPs in the absence of IL-6 and MgCl₂ (curve 1), in the presence of 8 mM MgCl₂ (curve 2), and in the presence of 250 pg per mL IL-6 and 8 mM MgCl₂ (curve 3). (inset) The corresponding photographs of the colorimetric response. TEM images of (b) the polyA-Apt@AuNPs, (c) severely aggregated polyA-Apt@AuNPs, and (d) less-aggregated polyA-Apt@AuNPs.

3.3 Optimization of experimental conditions

Aptamers prefer to adopt a folded conformation when binding to IL-6.³⁸ Incubation and reaction conditions, such as pH value and temperature, have impacts on target recognition. To achieve higher detection sensitivity, a series of control experiments were performed to optimize the incubation and reaction conditions. The differential absorbance ratio Δ(A₆₅₀/A₅₂₀) = (A₆₅₀/A₅₂₀)_{control −} (A₆₅₀/A₅₂₀)_sample, where (A₆₅₀/A₅₂₀)_control and (A₆₅₀/A₅₂₀)_sample are absorbance ratios at 650 nm and 520 nm measured at blank and solution with IL-6, respectively, used to characterize the effect of each parameter. The concentration of MgCl₂ was first investigated, as shown in Fig. 4a. The signal of Δ(A₆₅₀/A₅₂₀) experienced a significant increase as the concentration of MgCl₂ increased from 6 mM to 10 mM and then declined sharply. At low concentrations of MgCl₂, steric hindrance provided by polyA-Apt@AuNP binding with the target plays a significant role in the aggregation, while polyA-Apt@AuNPs without binding the target are susceptible to the counterions and undergo aggregation, resulting in a large margin of Δ(A₆₅₀/A₅₂₀). Once the concentration of MgCl₂ surpassed 10 mM, the folded aptamer may collapse due to the high salt concentration, leading to a net attractive van der Waals force and aggregation of polyA-Apt@AuNPs regardless of folded and unfolded aptamers. Ultimately, 10 mM MgCl₂ was selected as the optimum concentration of MgCl₂. The isoelectric point (pI) of the IL-6 protein is reported to be 6.96.³⁹ At pH values lower or higher than pI, the protein has a net positive or negative charge, respectively. Then, the pH of the PBS buffer was investigated, as shown in Fig. 4b. Increasing the pH from 5 to 6.5 improved the binding affinity between the aptamer and protein, making it the optimal choice. At pH higher than 6.5, the negative charge of the protein hindered their binding activity and lowered the signal. Then, a pH of 6.5 provided the best response. To determine the incubation time and temperature, incubation times ranging from 30 to 180 min and 20 to 42 °C were evaluated, respectively (Fig. 4c and d). These results indicated that the incubation of polyA-Apt@AuNPs and IL-6 at 37 °C for 90 min was adequate to generate a good signal. Then, the optimum conditions (10 mM MgCl₂, pH of PBS buffer 6.5, 90 min incubation time, and 37 °C incubation temperature) were determined for subsequent examinations.

Fig. 4 A ₆₅₀/A₅₂₀ and their differences measured by the polyA-Apt@AuNPs without and with IL-6. The optimization of the (a) MgCl₂ concentration, (b) pH of PBS buffer, (c) incubation time and (d) incubation temperature. Yellow columns: without IL-6 and pink columns: with 125 pg per mL IL-6. Red lines represent Δ(A₆₅₀/A₅₂₀) under different conditions. The error bars represent the standard deviations of the five measurements.

3.4 Analytical performance of polyA-Apt@AuNPs toward IL-6 detection

To assess the performance and sensitivity of the polyA-Apt@AuNPs for IL-6 detection, a series of artificial IL-6 samples ranging from 7 to 1000 pg mL⁻¹ were prepared for measurement. As shown in Fig. 5a, the intensity of the resonance wavelength at 650 nm decreased continuously with increasing concentrations of IL-6. The regression equation of A₆₅₀/A₅₂₀ = 1.368 − 0.325 × log₁₀[C_IL-6] and a determination coefficient of R² = 0.9863 confirmed a strong linear relationship between the logarithm of IL-6 concentrations and the absorption ratio ranging from 7 to 500 pg mL⁻¹. The limit of detection of this sensor using the commonly accepted method 3σ/S, where σ is the standard deviation of the blank sample signal and S is the slope of the calibration curve, was estimated to be 4.652 pg mL⁻¹. A comparison of the performance of this proposed sensor with that of other studies is illustrated in Table 1. The acceptable detection range and the LOD indicate its good performance in terms of cost and easy operation.

Fig. 5 Detection performance of the as-prepared polyA-Apt@AuNP aptasensor for IL-6 detection. (a) Absorption spectra response for the IL-6 detection in the range of 0–1000 pg mL⁻¹ and (b) calibration curve for the IL-6 detection in the range of 7–500 pg mL⁻¹ as logarithm values. The linear regression equation is A₆₅₀/A₅₂₀ = 1.368 − 0.325 × log₁₀[C_{IL_6}]. Each error bar represents the standard deviation across five replicate experiments.

Table 1 Comparison of the performance of different sensors for the detection of interleukin-6

Sensor	Material	Linear range	Detection limit (pg mL^−1)	Reference
Electrochemical immunosensor	CuNS	0.05–500 pg mL^−1	0.02	40
Amperometry	CDIa/streptavidin/IDEb	0.021–2100 pg mL^−1	0.021	41
Electrochemical magnetoimmuno sensor	Magnetic particles	1 pg mL^−1–1 µg mL^−1	0.3	42
Surface-enhanced Raman scattering	Au@MBN@Ag NPs	1–10^4 pg mL^−1	0.056	43
Localized surface plasmon resonance imaging (LSPRi) immunoassay	AuNPs	4.6–1 000 000 pg mL^−1	4.6	44
Colorimetry	AuNPs	3.3–125 µg mL^−1	1.95 × 10^6	29
Colorimetry	AuNPs	1–25 ng L^−1	70	31
Colorimetry	AuNPs	7–500 pg mL^−1	4.652	This work

---

To evaluate the specificity of the selectivity of the biosensor toward IL-6, its performance was compared using the described sensing procedure at the concentration of 250 pg mL⁻¹ for IL-6 and its analogues, including IL-4, IL-8 and IL-10. The obtained results are presented in Fig. 6. The value of A₆₅₀/A₅₂₀ for the target protein is approximately 37% of that of non-target proteins, which is much smaller compared to the signal generated by the non-target protein (the average values and relative standard deviations with five replicate analyses are shown in Table S2). This significant difference in response between IL-6 and other proteins indicates that this proposed sensing method possesses good performance in discriminating IL-6 against other interfering proteins.

Fig. 6 Selectivity study of the proposed aptasensor for 4 proteins. (a) Absorbance spectra for proteins at a concentration of 250 pg mL⁻¹. (b) The response of A₆₅₀/A₅₂₀ for each protein at a concentration of 250 pg mL⁻¹. Each error bar represents the standard deviation across five replicate experiments.

The response of this sensing strategy to IL-6 in the complex matrix, taking human serum, was further studied. IL-6 in serum with concentrations of 125, 250 and 500 pg mL⁻¹ was prepared, and the same detection step was carried out. The corresponding recovery rate is between 88.34% and 103.45% (Table 2), which shows that this method has a practical application value.

Table 2 Analytical results of interleukin 6 in serum samples using colorimetric sensors^a

Serum sample	Added (pg mL^−1)	Measured (pg mL^−1)	RSD (%, n = 5)	Recovery (%)
a RSD: relative standard deviation.
1	125	110.43	5.57	88.34
2	250	240.63	3.81	96.25
3	500	517.25	6.93	103.45

---

A commercially available human ELISA kit was also employed to quantify different concentrations of IL-6 for comparison. As shown in Fig. 7, the coefficient of determination using both methods for the spiked serum sample was 0.993, which demonstrates that the proposed colorimetric sensor possesses high accuracy in the determination of IL-6 in biological samples.

Fig. 7 Linear correlation between this proposed aptasensor and a commercially available ELISA kit for the detection of IL-6 spiked in serum. Each error bar represents the standard deviation across five replicate experiments.

4. Conclusions

Considering the critical importance of IL-6 as an inflammatory biomarker in relation to the risk of sepsis, coronary heart disease, metastatic melanoma, cardiovascular disease, etc., there is a growing demand for efficient, cost-effective and user-friendly methods for the detection of IL-6. The current study presents the concept of a colorimetric aptasensor platform for the sensitive and selective detection of IL-6. This detection method employs the steric hindrance provided by the folded aptamer to adjust the van der Waals force between AuNPs and render AuNPs transition from stability to aggregation under salt conditions, which is proportional to the concentration of IL-6. By evaluating the absorption ratio A₆₅₀/A₅₂₀, a linear range of 7–500 pg mL⁻¹ and 4.652 pg mL⁻¹ of LOD was determined. Furthermore, the selectivity and practicability of this biosensor have been demonstrated and shown to have potential in medical applications. In conclusion, this aptamer-functionalized AuNP-based biosensor provides a reliable method for detecting IL-6. Its low-cost and simplicity approach could open a new avenue to probe disease-related biomarkers with high sensitivity and specificity.

Author contributions

Yu Huang: conceptualization, writing – original draft, funding acquisition, Rui Liu: investigation, formal analysis, Chin-Jung Chuang: software, validation; Linhao Jiang: visualization, validation; Qiongyuan Zhang: data curation, validation; Jiangling Wu: methodology, supervision; Zhiguo Wu: data curation, software; Weiguang Yang: data curation; Qianye Zhang: software; Yuyan Sun: investigation; Ling Pan: formal analysis; Dongmei Liu: resources, project administration, writing – original draft; Yue Li: resources, project administration, writing – original draft, funding acquisition; Xing Chen: methodology; Lei Feng: supervision; Hua Zhang: investigation and Kaiji Xie: data curation.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data for this article, including Excel files containing absorption spectra and data for parameters optimization, are available at https://pan.baidu.com/s/1OIEqoKYEXt8ZIBfrM1vtYg?pwd=1234.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d5ay01582k.

Acknowledgements

This work was supported by the Special Cooperation Project of the Chinese Academy of Sciences and Hubei Province in 2024 and by the Natural Science Foundation of Chongqing (Grant No. CSTB2022NSCQ-MSX0060, CSTB2023NSCQ-MSX0175, and CSTB2025NSCQ-GPX0566).

References

1. G. Y. F. Ho, X. N. Xue, R. D. Burk, R. C. Kaplan, E. Cornell and M. Cushman, Variability of serum levels of tumor necrosis factor-alpha, interleukin 6, and soluble interleukin 6 receptor over 2 years in young women, Cytokine, 2005, 30(1), 1–6.
2. P. M. Ridker and M. Rane, Interleukin-6 Signaling and Anti-Interleukin-6 Therapeutics in Cardiovascular Disease, Circ. Res., 2021, 128(11), 1728–1746.
3. A. R. Blaser, J. Starkopf, M. Björck, A. Forbes, K. Kase, E. Kiisk, K. T. Laisaar, V. Mihnovits, M. Murruste, M. Maendul, A. L. Voomets and K. Tamme, Diagnostic accuracy of biomarkers to detect acute mesenteric ischaemia in adult patients: a systematic review and meta-analysis, World J. Emerg. Surg., 2023, 18(1), 44.
4. R. G. Kumar, M. L. Diamond, J. A. Boles, R. P. Berger, S. A. Tisherman, P. M. Kochanek and A. K. Wagner, Acute CSF interleukin-6 trajectories after TBI: Associations with neuroinflammation, polytrauma, and outcome, Brain Behav. Immun., 2015, 45, 253–262.
5. F. Kawasaki, Y. Kawano, Z. K. Hasan, H. Narahara and I. Miyakawa, The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids, Clin. Exp. Med., 2003, 3(1), 27–31.
6. O. J. McElvaney, G. F. Curley, S. Rose-John and N. G. McElvaney, Interleukin-6: obstacles to targeting a complex cytokine in critical illness, Lancet Respir. Med., 2021, 9(6), 643–654.
7. D. M. Franco, I. Arevalo-Rodriguez, M. R. I. Figuls, N. G. M. Oleas, X. Nuvials and J. Zamora, Plasma interleukin-6 concentration for the diagnosis of sepsis in critically ill adults, Cochrane Database Syst. Rev., 2019,(4), Cd011811.
8. L. Hoejberg, L. Bastholt, J. S. Johansen, I. J. Christensen, J. Gehl and H. Schmidt, Serum interleukin-6 as a prognostic biomarker in patients with metastatic melanoma, Melanoma Res., 2012, 22(4), 287–293.
9. D. K. Thompson, K. M. Huffman, W. E. Kraus and V. B. Kraus, Critical Appraisal of Four IL-6 Immunoassays, PLoS One, 2012, 7(2), e30659.
10. A. Alba-Patiño, S. M. Russell, M. Borges, N. Pazos-Pérez, R. A. Alvarez-Puebla and R. de la Rica, Nanoparticle-based mobile biosensors for the rapid detection of sepsis biomarkers in whole blood, Nanoscale Adv., 2020, 2(3), 1253–1260.
11. M. Tertis, P. I. Leva, D. Bogdan, M. Suciu, F. Graur and C. Cristea, Impedimetric aptasensor for the label-free and selective detection of Interleukin-6 for colorectal cancer screening, Biosens. Bioelectron., 2019, 137, 123–132.
12. N. Nesakumar, S. Kesavan, C. Z. Li and S. Alwarappan, Microfluidic Electrochemical Devices for Biosensing, J. Anal. Test., 2019, 3(1), 3–18.
13. M. Muhammad, C. S. Shao and Q. Huang, Aptamer-functionalized Au nanoparticles array as the effective SERS biosensor for label-free detection of interleukin-6 in serum, Sens. Actuators, B, 2021, 334, 129607.
14. T. Zhou, D. C. Lu, Q. T. She, C. R. Chen, J. B. Chen, Z. F. Huang, S. Y. Feng, R. Y. You and Y. D. Lu, Hypersensitive detection of IL-6 on SERS substrate calibrated by dual model, Sens. Actuators, B, 2021, 336, 129597.
15. M. Majdinasab, A. Azziz, Q. Q. Liu, V. Mora-Sanz, N. Briz, M. Edely and M. L. de la Chapellea, Label-free SERS for rapid identification of interleukin 6 based on intrinsic SERS fingerprint of antibody-gold nanoparticles conjugate, Int. J. Biol. Macromol., 2023, 253, 127560.
16. M. Tertis, B. Ciui, M. Suciu, R. Sandulescu and C. Cristea, Label-free electrochemical aptasensor based on gold and polypyrrole nanoparticles for interleukin 6 detection, Electrochim. Acta, 2017, 258, 1208–1218.
17. B. Fan, S. P. Wang, S. S. Huang, S. Ma, H. C. Zuo, S. Li and Z. T. Chen, Real-time Monitoring of Wound Infection Based on Multi-channel Biomedical Sensors, J. Anal. Test., 2025 DOI:10.1007/s41664-025-00391-w.
18. W. T. Ting, M. J. Wang and M. M. R. Howlader, Interleukin-6 electrochemical sensor using poly (o-phenylenediamine)-based molecularly imprinted polymer, Sens. Actuators, B, 2024, 404, 135282.
19. M. Mahani, M. Faghihi-Fard, F. Divsar, M. Torkzadeh-Mahani and F. Khakbaz, Ultrasensitive FRET-based aptasensor for interleukin-6 as a biomarker for COVID-19 progression using nitrogen-doped carbon quantum dots and gold nanoparticles, Microchim. Acta, 2022, 189(12), 472.
20. Y. Wu, T. T. Jiang, Z. Y. Wu and R. Q. Yu, Internal standard-based SERS aptasensor for ultrasensitive quantitative detection of Ag+ ion, Talanta, 2018, 185, 30–36.
21. Y. Huang, X. Y. Pu, H. S. Qian, C. J. Chuang, S. S. Dong, J. L. Wu, J. J. Xue, W. Cheng, S. J. Ding and S. Q. Li, Optical fiber surface plasmon resonance sensor using electroless-plated gold film for thrombin detection, Anal. Bioanal. Chem., 2024, 416(6), 1469–1483.
22. M. Sabela, S. Balme, M. Bechelany, J. M. Janot and K. Bisetty, A Review of Gold and Silver Nanoparticle-Based Colorimetric Sensing Assays, Adv. Eng. Mater., 2017, 19(12), 1700270.
23. Y. J. Song, W. L. Wei and X. G. Qu, Colorimetric Biosensing Using Smart Materials, Adv. Mater., 2011, 23(37), 4215–4236.
24. J. Y. Byun, Y. B. Shin, D. M. Kim and M. G. Kim, A colorimetric homogeneous immunoassay system for the C-reactive protein, Analyst, 2013, 138(5), 1538–1543.
25. G. Q. Wang, Y. Q. Wang, L. X. Chen and J. Choo, Nanomaterial-assisted aptamers for optical sensing, Biosens. Bioelectron., 2010, 25(8), 1859–1868.
26. J. Oishi, Y. Asami, T. Mori, J. H. Kang, T. Niidome and Y. Katayama, Colorimetric enzymatic activity assay based on noncrosslinking aggregation of gold nanoparticles induced by adsorption of substrate peptides, Biomacromolecules, 2008, 9(9), 2301–2308.
27. W. Zhao, M. A. Brook and Y. F. Li, Design of Gold Nanoparticle-Based Colorimetric Biosensing Assays, Chembiochem., 2008, 9(15), 2363–2371.
28. W. A. Zhao, W. Chiuman, J. C. F. Lam, S. A. McManus, W. Chen, Y. G. Cui, R. Pelton, M. A. Brook and Y. F. Li, DNA aptamer folding on gold nanoparticles: From colloid chemistry to biosensors, J. Am. Chem. Soc., 2008, 130(11), 3610–3618.
29. S. Giorgi-Coll, M. J. Marín, O. Sule, P. J. Hutchinson and K. L. H. Carpenter, Aptamer-modified gold nanoparticles for rapid aggregation-based detection of inflammation: an optical assay for interleukin-6, Microchim. Acta, 2020, 187(1), 13.
30. A. L. Chen, M. M. Yan and S. M. Yang, Split aptamers and their applications in sandwich aptasensors, TrAC, Trends Anal. Chem., 2016, 80, 581–593.
31. M. Malekmohamadi, S. Mirzaei, A. H. Rezayan, V. Abbasi and A. A. Mehrizi, µPAD-based colorimetric nanogold aptasensor for CRP and IL-6 detection as sepsis biomarkers, Microchem. J., 2024, 197, 109744.
32. J. Liu and Y. Lu, Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes, Nat. Protoc., 2006, 1(1), 246–252.
33. K. M. Koo, A. A. I. Sina, L. G. Carrascosa, M. J. A. Shiddiky and M. Trau, DNA-bare gold affinity interactions: mechanism and applications in biosensing, Anal. Methods, 2015, 7(17), 7042–7054.
34. H. Pei, F. Li, Y. Wan, M. Wei, H. J. Liu, Y. Su, N. Chen, Q. Huang and C. H. Fan, Designed Diblock Oligonucleotide for the Synthesis of Spatially Isolated and Highly Hybridizable Functionalization of DNA-Gold Nanoparticle Nanoconjugates, J. Am. Chem. Soc., 2012, 134(29), 11876–11879.
35. Y. Huang, S. Q. Li, C. Y. Liu, L. G. Chen, H. S. Qian, H. P. Ho, J. L. Wu, J. Wu and X. Y. Pu, One-step competitive assay for detection of thrombin via disassembly of diblock oligonucleotide functionalised nanogold aggregates, Sens. Actuators, B, 2023, 376, 133032.
36. Y. Y. Xie, Y. Huang, D. Y. Tang, H. L. Cui and H. Y. Cao, A competitive colorimetric chloramphenicol assay based on the non-cross-linking deaggregation of gold nanoparticles coated with apolyadenine-modified aptamer, Microchim. Acta, 2018, 185(12), 534.
37. Y. Y. Xie, Y. Huang, J. Y. Li and J. L. Wu, A trigger-based aggregation of aptamer-functionalized gold nanoparticles for colorimetry: An example on detection of Escherichia coli O157:H7, Sens. Actuators, B, 2021, 339, 129865.
38. K. L. Rhinehardt, S. A. Vance, R. V. Mohan, M. Sandros and G. Srinivas, Molecular modeling and SPRi investigations of interleukin 6 (IL6) protein and DNA aptamers, J. Biomol. Struct. Dyn., 2018, 36(8), 1934–1947.
39. M. T. Hwang, I. Park, M. Heiranian, A. Taqieddin, S. Y. You, V. Faramarzi, A. A. Pak, A. M. Zande, N. R. Aluru and R. Bashir, Ultrasensitive Detection of Dopamine, IL-6 and SARS-CoV-2 Proteins on Crumpled Graphene FET Biosensor, Adv. Mater. Technol., 2021, 6(11), 2100712.
40. W. Jesadabundit, S. Jampasa, R. D. Crapnell, N. C. Dempsey, C. E. Banks, W. Siangproh and O. Chailapakul, Toward the rapid diagnosis of sepsis: dendritic copper nanostructure functionalized diazonium salt modified screen-printed graphene electrode for IL-6 detection, Microchim. Acta, 2023, 190(9), 362.
41. N. Chen, H. Yang, Q. Li, L. J. Song, S. C. B. Gopinath and D. Wu, An interdigitated aptasensor to detect interleukin-6 for diagnosing rheumatoid arthritis in serum, Biotechnol. Appl. Biochem., 2021, 68(6), 1479–1485.
42. M. Tertis, G. Melinte, B. C. Ciui, I. Simon, R. Stiufiuc, R. Sandulescu and C. Cristea, A Novel Label Free Electrochemical Magnetoimmunosensor for Human Interleukin-6 Quantification in Serum, Electroanalysis, 2019, 31(2), 282–292.
43. Q. Huang, X. Chen, M. Fan, S. Y. Ruan, S. R. Peng, R. Y. You, J. B. Chen and Y. D. Lu, SERS-based self-calibrating aptamer sensor for selective detection of IL-6, Sens. Actuators, B, 2023, 374, 132828.
44. J. C. He, L. Zhou, G. T. Huang, J. L. Shen, W. Chen, C. Y. Wang, A. Kim, Z. Y. Zhang, W. Q. Cheng, S. Y. Dai, P. Y. Chen and F. Ding, Enhanced Label-Free Nanoplasmonic Cytokine Detection in SARS-CoV-2 Induced Inflammation Using Rationally Designed Peptide Aptamer, ACS Appl. Mater. Interfaces, 2022, 14(43), 48464–48475.

---

Footnote

† These authors contributed equally to this work.

---