A label-free fluorescent aptasensor for sensitive detection of pb2+ ions using hollow gold nanoparticles

Abstract

A label-free turn-on fluorescent aptasensor for sensitive detection of Pb²⁺ ions is disclosed. The sensor employs hollow gold nanoparticles (HGNPs) as nanocarriers, a Pb²⁺-specific aptamer as the biorecognition element, rhodamine B (RDB) as a fluorescent dye, and a chitosan (CTS) coating to prevent premature dye leakage. In the absence of Pb²⁺, the aptamer binds to HGNPs and CTS stabilizes the nanocomplex, resulting in weak fluorescence. Upon Pb²⁺ introduction, the aptamer undergoes conformational changes, releasing RDB from the HGNP cavity and producing a strong fluorescence signal. The aptasensor demonstrates high sensitivity with a detection limit of 1 ppb and excellent specificity over other metal ions. It enables reliable quantification of Pb²⁺ in environmental and biological samples, offering a cost-effective and user-friendly alternative to conventional analytical methods.

Description

[0001] A label-free fluorescent aptasensor for sensitive detection of Pb2+ions using hollow gold nanoparticles

[0002] Technical Field:

[0003] Environmental pollution, particularly from toxic heavy metals, has emerged as a critical global challenge requiring effective management Among these, lead (II) ion (Pb2+) is considered highly hazardous due to its resistance to degradation and its capacity for human absorption. It poses severe health risks, notably to the nervous system and kidneys, and can lead to fatal outcomes. The World Health Organization (WHO) recommends a maximum allowable concentration of Pb2+ in drinking water at 10 parts per billion (ppb), a standard echoed by the International Agency for Research on Cancer (IARC), while the U.S. Environmental Protection Agency allows up to 15 ppb. To mitigate the adverse impacts of toxic ions on the environment and human health, effective detection methods are essential. Traditional techniques such as inductively coupled plasma atomic emission spectroscopy (ICP-AES), flame atomic absorption spectrometry (FAAS), inductively coupled plasma-mass spectrometry (ICP-MS), and high-performance liquid chromatography (HPLC) require complex sample preparation and expensive equipment. Hence, there is a pressing need for more economical, sensitive, and straightforward detection alternatives. Fluorescence-based biosensors have emerged as a promising solution due to their robust detection capabilities and versatility, allowing for the identification of various target molecules, including pathogens, food contaminants, and metal ions.

[0004] Label-free biosensors have attracted considerable attention as a viable alternative to traditional label-based assays due to their cost-effectiveness, ease of use, user-friendliness, and durability. Moreover, these biosensors are particularly advantageous for detecting analytes and target molecules that are difficult or impossible to label. In our invention, we present a turn-on fluorescent aptasensor specifically designed to detect Pb2+. This sensor employs hollow gold nanoparticles (HGNPs) as a carrier, together with a fluorescent dye and a label-free aptamer. The interaction between the aptamer and Pb2+leads to structural changes in the nanocomplex, resulting in the release of the dye and an increased fluorescent signal. The HGNPs-based aptasensor offers a simple, cost-effective, and reliable method for quantifying Pb2+, with significant potential for applications in environmental and biological monitoring.Background:

[0005] Lead contamination represents a major global health and environmental challenge due to its high toxicity, bioaccumulation, and resistance to degradation. Even at very low concentrations, Pb2+can cause severe damage to the nervous system, kidneys, and other organs. Regulatory agencies, including the World Health Organization (WHO) and the U.S. Environmental Protection Agency (EP A), have set strict limits on Pb2+levels in drinking water, underscoring the urgent need for reliable monitoring technologies.

[0006] Conventional detection techniques such as ICP-MS, FAAS, and HPLC offer high sensitivity but are limited by their complexity, cost, and need for sophisticated instrumentation. Fluorescencebased biosensors, and in particular label-free aptasensors, have emerged as promising alternatives because they combine simplicity, low cost, and high sensitivity. However, there remains a need for improved approaches that provide enhanced stability, reproducibility, and ease of use in real- world environmental applications.

[0007] Summary:

[0008] A sensitive fluorescent aptasensor was developed for lead ion (Pb2+) detection using hollow gold nanoparticles (HGNPs) as a nano-carrier and rhodamine B (RDB) as a fluorescent signal agent. The aptasensor operates through the specific binding of aptamers to Pb2+, resulting in the release of RDB from chitosan-coated HGNPs, thereby enhancing fluorescence intensity( Fig.l). This method achieved a detection limit of 1 ppb and a linear dynamic range of 2 to 1000 ppb. It demonstrated effectiveness in measuring Pb2+ concentrations in human serum, low-fat milk, and mineral water. The proposed biosensor is characterized by simplicity, user-friendliness, costeffectiveness, and high sensitivity, making it suitable for complex environmental and clinical sample analysis.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS:

[0010] Fig 1. Illustrates the mechanism of turn-on fluorescent aptasensor for detecting Pb2+utilizing HGNPs, chitosan, rhodamine B, and a label-free aptamer.

[0011] Fig 2. Illustrates the Size and morphological features of the HGNPs (A) and Apt-CTS-HGNPs@RDB NPs (B) revealed by SEM.Fig 3. Illustrates the fluorescence intensity of the Apt-CTS-HGNPs@RDB when exposed to varying quantities of CTS / HGNPs over different incubation periods. (B) The fluorescence intensity of the Apt-CTS-HGNPs@RDB nanocomplex in the presence of Pb2+(20 ppb) over different incubation periods (n =3).

[0012] Fig 4. Illustrates the calibration curve of the developed method when exposed to various amounts of Pb2+ (n =3). The linear relationship between the relative fluorescence intensity and the various concentrations of Pb2+ (2 ppb-1000 ppb), obtained under the optimal conditions (CTS / HGNPs: 12:1, CTS incubation time: 24 h, Pb2+ incubation time: 15 min). The error bars represent the standard deviation for three replicates.

[0013] Fig 5. Illustrates Selectivity of the approach toward Pb2+(30 ppb) with the other ionic compounds at 100 ppb (n =3). Fluorescence intensity of samples containing other ionic compounds were performed under optimal conditions. The error bars represent the standard deviation (n =3).

[0014] Fig 6. Illustrates the results of recovery test of the turn-on fluorescent aptasensor and atomic absorption spectroscopy in water, human serum, and milk sample.

[0015] DETAILED DESCRIPTION

[0016] • In the present study, a turn-on fluorescent aptasensor for monitoring Pb2+ was fabricated using HGNPs as nanocarrier, aptamer as a biorecognition element, RDB as a cost-effective fluorescent dye, and CTS to prevent RDB leakage in the absence of target. The detection mechanism of the proposed aptasensor is based on the specific binding of aptamer with Pb2+and leakage of RDB dye from the internal cavity of HGNPs, as well as the difference in fluorescence intensity of RDB dye in the loaded state in the HGNPs cavity and the free state (leakage into the environment). In the absence of Pb2+, the specific aptamer was attached onto the surface of HGNPs. Then, CTS was coated on the HGNPs surface and prevents the leakage of RDB, in which case, a weak fluorescence intensity was observed. Upon introduction of Pb2+into the solution, the aptamer’s interaction with it led to the release of RDB from the HGNPs ’cavity, resulting in an increase in fluorescence intensity. The rise in fluorescence intensity of the developed aptasensor was directly correlated with the concentration of Pb2+.

[0017] • Hollow gold nanoparticles (HGNPs) were synthesized through a process involving the reduction of gold salt on cobalt nanoparticles. Initially, sodium borohydride was rapidly injectedinto a mixture of sodium citrate and cobalt chloride under an argon atmosphere after ultrasonication to remove gas. This led to the creation of cobalt boride nanoparticles (CoB NPs), evidenced by the resultant brown solution. The subsequent addition of HAuC14 facilitated anaerobic galvanic replacement, resulting in a blue-green solution characteristic of HGNPs. This study employed SEM imaging techniques to analyze the structure of HGNPs and the Apt-CTS-HGNPs@RDB complex, revealing a circular morphology and uniform distribution for both samples. Additionally, DLS measurements were conducted to ascertain the average hydrodynamic diameter and zeta potential of various formulations, including HGNPs, HGNPs@RDB, Apt-HGNPs@RDB, and Apt-CTS-HGNPs@RDB, thereby demonstrating the effectiveness of the current investigation (Fig.2).

[0018] The average size of HGNPs was found to be 61.25 ± 2.18 nm, which increased to 74.37 ± 5.81 nm after the formation of Apt-HGNPs@RDB (Table.1). The addition of a chitosan (CTS) layer further expanded the size to 91.35 ± 3.51 nm for Apt-CTS-HGNPs@RDB. Zeta potential measurements indicated values of -31.3 mV for HGNPs and -29.8 mV for Apt-HGNPs@RDB, while Apt-CTS-HGNPs@RDB measured -19.4 mV. The difference in zeta potential between HGNPs and Apt-CTS-HGNPs@RDB is attributed to the chitosan layer, which exhibits a lower negative charge.

[0019] Table.1. Zeta potential and size of the HGNPs, HGNPs@RDB, Apt-HGNPs@RDB and Apt-CTS- HGNPs@RDB NPs.

[0020]

[0021] Sensing approach: Sensing approach was developed by combining 2 m of HGNPs with 10 pL of RDB (1 mM) and allowing the mixture to react for 24 hours. Following this, 1 pL of lead aptamer (30 pM) and 10 pL of Tween 80 (10% v / v) were added, and the solution was reacted for an additional 4 hours. To prevent dye leakage from the nanocomplex, a layer of chitosan (CTS) was applied using 0.5 mg of chitosan powder mixed with 500 pL of dry ether, incubated for 24 hours. Subsequently, 50 pL of Pb2+ (ranging from 2-1000 ppb) was introduced to the solution and reacted for 15 minutes, during which the fluorescence intensity was monitored at excitation and emission wavelengths of 553 nm and 574 nm, respectively.• The agarose gel retardation assay was performed to confirm the binding of the aptamer to HGNPs. The results, illustrated in Fig.3, showed that the unbound aptamer band was absent in lane C (Apt- CTS-HGNPs@RDB), indicating effective attachment to the nanoparticles. Additionally, the absence of residual aptamer in the filtrate from the Apt-CTS HGNPs@RDB solution further confirms the successful binding.

[0022] • To effectively prevent the leakage of RDB from nanoparticles, a coating of chitosan (CTS) is necessary, ensuring that RDB is released only upon aptamer binding to Pb2+ ions. Various CTS to hyperbranched gold nanoparticles (HGNPs) ratios were tested (1:3, 1:1, 3:1, 6:1, and 12:1), with a 12:1 ratio emerging as optimal due to its ability to minimize premature RDB leakage (Fig.4). This ratio exhibited a significantly lower leakage rate compared to others. Results demonstrated that after 24 hours, the fluorescence signal was minimal, confirming successful CTS coating and indicating that a 24-hour incubation period is optimal for the surface coverage of aptamer-modified HGNPs containing RDB.

[0023] • The fluorescence emission of RDB peaked after a 15 -minute exposure to Pb2+ at a concentration of 20 ppb, establishing this duration as optimal for RDB release from the complex (Fig.5). Subsequent observations indicated a decrease in RDB leakage over time, likely due to the reabsorption of positively charged RDB by the negatively charged HGNPs beyond the 15 -minute mark.

[0024] • Under optimized conditions, the turn-on fluorescent aptasensor demonstrated quantitative detection of Pb2+ with relative fluorescence intensities measured at 574 nm. The results indicate a linear increase in fluorescence intensity corresponding to logarithmic concentrations of Pb2+ ranging from 2 ppb to 1000 ppb. The limit of detection (LOD) for this aptasensor was determined to be 1 ppb, calculated using the IUPAC formula (3Sb / slope), where Fo and F represent fluorescence intensities at 574 nm without and with Pb2+, respectively (Fig.6).

[0025] • The specificity of a biosensor is critical for its effectiveness in distinguishing between various ions.

[0026] Results from specificity tests demonstrate a significantly stronger response to Pb2+ compared to other ions, including Mg2+, Fe3+, Ca2+, Na+, K+, Al3+, Ars3+, Hg2+, Zn2+, Cd2+, Ag+, and Cu2+(Fig.7). This finding not only confirms the aptasensor's excellent specificity but also emphasizes its rapid performance, marking it as a key advantage.• The performance of an aptasensor for detecting Pb2+ was evaluated using human serum, mineral water, and low-fat milk samples. Different concentrations of Pb2+ were spiked in diluted samples, followed by filtration to remove suspended particles (Table.2). The aptasensor achieved recovery rates between 99.8% and 110.8%, indicating its effective functionality for analyzing complex matrices. Additionally, comparisons with atomic absorption spectrometry demonstrated a strong correlation between the results obtained from the fluorescent aptasensor and the conventional method, highlighting the aptasensor's reliability for quantitative analysis.

Claims

ClaimsWhat is claimed is:

1. An aptasensor for detecting Pb2+ions in a sample, comprising:• a nanoparticle carrier;• a fluorescent dye associated with the nanoparticle carrier; and• a Pb2+-specific aptamer configured to selectively bind Pb2+ions.

2. The aptasensor of claim 1, wherein the nanoparticle carrier comprises hollow gold nanoparticles (HGNPs).

3. The aptasensor of claim 1 and 2, wherein the fluorescent dye comprises rhodamine B encapsulated inside the cavity of the nanoparticles.

4. The aptasensor of claim 1 , wherein a stabilizing polymer coating covers at least a portion of the nanoparticle carrier to prevent premature leakage of the fluorescent dye.

5. The aptasensor of claim 4, wherein the stabilizing polymer comprises chitosan.

7. The aptasensor of claim 1 and 4, wherein upon exposure to Pb2+ions, the aptamer and stabilizing polymer are released, thereby triggering the release of the fluorescent dye from the nanoparticles.

6. The aptasensor of claim 1, wherein the aptamer is electrostatically attached to the nanoparticle carrier to enable selective recognition of Pb2+ions.

8. The aptasensor of claim 1, wherein the aptasensor provides a turn-on fluorescence response upon binding of Pb2+ions.

9. The aptasensor of claim 1, wherein the aptasensor detects Pb2+ions at concentrations ranging from 2 to 1000 ppb with a detection limit of about 1 ppb.

10. The aptasensor of claim 1, wherein the aptasensor selectively detects Pb2+ions compared to other metal ions including Fe3+, K+, Na+, Mg2+, Ca2+, Al3+, As3+, Hg2+, Zn2+, Cd2+, Ag+, and Cu2+.

11. The aptasensor of claim 1, wherein the aptasensor is applied for detection of Pb2+ions in real samples selected from drinking water, milk, and human serum.

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