The potential toxicity of environmental pollutants and pharmaceutical candidates to human health relates to their metabolites reactive oxygen species (ROS) [1
]. They cause the damage of genetic materials, which refers to genotoxicity, giving rise to carcinogenesis, diabetes, aging, neurodegenerative, and cardiovascular diseases. Guanine has the lowest oxidation potential among the four nucleobases of the DNA molecule, and it is most susceptible to oxidative damage [3
]. In particular, 8-hydroxy-2′-deoxyguanosine (8-OHdG) is an oxidized product of the deoxyguanosine residues in DNA, which is formed from the attack of hydroxyl radicals (•OH) at the C-8 position of guanine [4
]. Thus, 8-OHdG molecules are excreted into urine. The urinary 8-OHdG reflects the extent of oxidative DNA damage in human body [5
]. Hence, it has an impact on analytical science to develop a sensitive and selective platform for 8-OHdG determination and evaluation.
A variety of analytical methods, such as resonance Rayleigh scattering (RRS), high-performance liquid chromatography with electrochemical detection (HPLC-ED), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis (CE), and enzyme-linked immunosorbent assay (ELISA), have been developed for 8-OHdG investigation [6
]. These methods can determine 8-OHdG with good selectivity and suitable detection limit, but most of them suffer from expensive instrument, high requirement of sample pretreatment, and operation processes. Some biosensor devices based on electrochemical or electrochemiluminescence detection modes have been developed for 8-OHdG detection [10
]. Importantly, 8-OHdG can be directly read making use of active materials or highly conductive materials of carbon nanotubes or graphene modified on electrodes. Electrochemical sensors have obvious advantages including low cost, easy miniaturization, and simplicity, but their performance is affected by material properties of electrodes [11
]. Fluorescence resonance energy transfer (FRET) technology has been served as the designing strategy of various biosensors for medical diagnosis, food safety, and environmental detection due to its high sensitivity, facile operation, and high specificity [12
]. A luminescent paper-based device and a fluorescence aptasensor were developed for oxidative stress biomarker 8-OHdG detection [13
]. The fluorescence signals relied on chemical fluorophore, which had the disadvantage of photobleaching.
Nanoscale metal−organic frameworks (nMOFs) emerge as an important class of nanomaterials in diverse fields because of their charming properties of high porosity, structural diversity, and multifunctionality. They have attracted much attention in areas of gas storage and separation, energy conversion, and sensing [15
]. However, it should be noted that the research of nMOFs in the sensing field should still be further explored deeply in the future. Zeolitic imidazolate framework-8 (ZIF-8) is a kind of the typical nMOFs materials [18
]. It is composed of ZnN4 tetrahedral structure unit formed by zinc ion (Zn2+
) and nitrogen atom (N) in methylimidazole ester. It has a zeolite structure with large pores and shows simple crystal structure, good stability, and high loading capacity. ZIF-8 nanocrystals are used as the host material. Both gold nanoparticles and silver nanoparticles can be used as guests to be encapsulated in ZIF-8 nanocrystals to form [email protected]
nanoparticles and [email protected]
nanoparticles, respectively [20
]. These structures avoid nanoparticles being disturbed by the environment. Compared to [email protected]
nanoparticles, the synthesis process of [email protected]
nanoparticles can be easily controlled and the size of gold nanoparticles dispersed on ZIF-8 crystals is uniform. [email protected]
nanoparticles have property synergies of inorganic nanoparticles and MOFs for multifunctional applications. They combine the advantages of structural adaptivity, flexibility, high specific surface area, and high fluorescence quenching efficiency [21
]. [email protected]
nanoparticles are candidates for highly sensitive molecule detection. Unsaturated metal site makes it easy to be modified for biosensing, targeted imaging, and therapy.
Since biosensor detection is mainly performed on solid substrate. Nanoporous alumina membranes are covered with through nanopores, which make the membranes have high surface-to-volume ratio property. Together with the advantages of easy fabrication and surface biofunctionalization, nanoporous membranes are good substrates for constructing biosensors to detect neurotoxin, nucleic acid, proteins, virus, and food hazards [23
]. Nanoporous alumina membrane-based FRET biosensors with nanoparticles as the fluorescence donors and acceptors provide easy operation, good photostability, and sensitivity for 8-OHdG detection. Traditional semiconducting quantum dots are toxic and cause environmental pollution due to the heavy metal components. Organic dyes as fluorescent probes can be easily obtained but suffer from photobleaching [29
]. Carbon dots (CDs) emerge as superior fluorophores due to their advantageous virtues, such as low cost, excellent photostability, nontoxicity, and ease to be modified with biomolecules [30
]. These advantageous merits make CDs attractive candidates in the areas of sensing and catalysis applications [32
]. CDs together with gold nanoparticles have been used to develop a FRET assay method for detecting DNA containing oxidatively damaged product 8-OHdG (DNA-8-OHdG) [38
]. CDs and gold nanoparticles acted as the donor and acceptor pair. The detection process was performed in the solution. CDs modified on nanoporous alumina membranes can form good substrates with fluorescence and extends the assay to the solid substrate for 8-OHdG biosensing.
In the study, we develop a sensitive FRET biosensor based on CDs-modified nanoporous alumina membrane with CDs as fluorescence donors and [email protected]
nanoparticles as signal quenchers for 8-OHdG detection in urine. Nanoporous alumina membranes were conjugated with CDs, which were modified with glutaraldehyde for 8-OHdG antibody conjugation. CDs-modified nanoporous alumina membranes were used as the biosensor substrate for 8-OHdG detection. [email protected]
nanoparticles were functionalized with 8-OHdG antibody. CDs and [email protected]
nanoparticles acted as the fluorescence donors and signal quenchers, respectively. The addition of 8-OHdG to the biosensor substrate can bring [email protected]
nanoparticles closely to CDs. The emission of CDs under 350 nm photoexcitation was quenched by [email protected]
nanoparticles leading to FRET effect on the nanoporous alumina membrane substrate. Further, 8-OHdG can be detected by calculating the fluorescence intensity change with FRET effect. The limit of detection (LOD) of this FRET biosensor for 8-OHdG detection is 0.31 nM. It shows the potential applications for sensitive DNA damage biomarker detection.
2. Materials and Methods
Specifically, 8-OHdG, bovine serum albumin (BSA), citric acid, diethylenetriamine (EDTA), (3-glycidyloxypropyl)trimethoxysilane (GPMS), glutaraldehyde, methylbenzene, chloroauric acid, 3-mercaptopropionic acid (MPA), zinc nitrate hexahydrate (Zn(NO3)2·6H2O), methanol (99.8%), sodium chloride, potassium chloride, calcium chloride, acetone, N,N-dimethylformamide (DMF), sodium borohydride (NaBH4, 99.99%), and dehydrated alcohol were obtained from Sigma Aldrich (St. Louis, Missouri (Mo), USA). NaCl, KCl, CaCl2, MgCl2, thymine, cytosine, adenine, guanine, and hydrogen peroxide were ordered from ALADDIN Reagent (Shanghai, China). Nanoporous alumina membranes were purchased from Whatman (Boston, Massachusetts (Ma), USA). Alongside, 8-OHdG antibody was bought from Abcam (Cambridge, UK). In addition, 2-methylimidazole (2-MeIM, 99%), 1-dodecanethiol (DDT, ≥98%), hexadecyltrimethyl ammonium bromide (CTAB, 98%), silver nitrate (AgNO3, ≥99.0%), 11-mercaptoundecanoic acid (MUA, 95%), and 4-nitrophenol (4-NP, ≥99.5%) were purchased from Sigma Aldrich (St. Louis, Missouri (Mo), USA). The chemicals were used as received without further purification.
2.2. Synthesis of Carbon Dots (CDs)
CDs were synthesized with citric acid and EDTA by one-step hydrothermal treatment [38
]. Citric acid (0.22 g) was dissolved in deionized water (DI water, 10 mL). EDTA (112.6 μL) was added to citric acid solution. The mixture was stirred thoroughly, transferred, and sealed into a clean and dry Teflon equipped stainless steel autoclave. Hydrothermal treatment at 200 °C for 6 h was carried out on it and then was cooled down naturally to room temperature. After centrifugation of dark brown solution at 13.200 rpm/min for 15 min, the supernatant was freeze-dried to collect CDs. CDs (1 mg) were dissolved in DI water (1 mL) and stored for later use.
2.3. Surface Functionalization of Nanoporous Alumina Membrane
Nanoporous alumina membranes, which had pore diameters of 200 nm, were surface functionalized according to the procedures in our previous studies [23
]. They were boiled in hydrogen peroxide for 30 min to form hydroxyl groups on the surface and rinsed by DI water. After gentle shaking for 15 min, nanoporous membranes were dried and immersed in solution of GPMS and methylbenzene (2%). They were sealed and reaction was carried out at 60 °C for 24 h. They were washed 3 times by acetone and absolute ethyl alcohol. The membranes were cured at 60 °C for 2 h and stored with desiccation. CDs solution (0.1 mg/mL, 150 μL) was added to the as-prepared silanized nanoporous alumina membranes, and kept overnight in moist and dark environment at room temperature. The membranes were rinsed in DI water. Glutaraldehyde (10 μL) was added to membranes for 30 min to activate amino group of CDs that were immobilized on membranes. After that, 8-OHdG antibody solution (1 μg/mL, 150 μL) was added to membranes and incubated overnight at 4 °C. Further, 8-OHdG antibody was conjugated on CDs with glutaraldehyde as linker. The functionalized nanoporous membranes were cleaned by three repeated wash cycles and stored at 4 °C.
nanoparticles were prepared according to previous study with slight modification [20
]. Chloroauric acid and MPA were dissolved in methanol solution with the concentrations of 0.01 and 0.1 M, respectively. Chloroauric acid solution (1.5 mL) and MPA solution (0.45 mL) were mixed, stirring for 3 h at room temperature. The formed Au-MPA compounds was collected and purified by centrifugation (10,000 rpm/min, 10 min) followed by washing with methanol twice. The precipitate was resuspended in methanol solution (4 mL). Zinc nitrate and 2-methyl imidazole were dissolved in DMF with the concentrations of 0.070 and 0.43 M, respectively. The zinc nitrate solution (4.8 mL) was added to the same volume of 2-methyl imidazole solution, stirring for 5 min. The mixture was pipetted and transferred to a dry Teflon equipped stainless steel autoclave (100 mL). The reaction lasted for 6 h at 120 °C. ZIF-8 was synthesized and collected by centrifugation (10,000 rpm/min, 10 min). It was washed by methanol twice and resuspended in 10 mL methanol solution. The as-prepared Au-MPA solution (2 mL) was diluted with methanol solution. The freshly prepared methanol solution of R-NBH4
(4 mL, 0.25 M) was added to Au-MPA solution, stirring for 15 min. Then, the as-prepared ZIF-8 solution (8 mL) was mixed with the above solution and stirred for 1 h to form [email protected]
nanoparticles. They were collected by centrifugation (10,000 rpm/min, 10 min) and washed twice by methanol. The precipitate was resuspended in 16 mL methanol solution for later use.
2.5. Surface Biofunctionalization of [email protected] Nanoparticles
nanoparticle solution was centrifuged with the speed of 8000 rpm/min for 10 min and rinsed twice by DI water. [email protected]
nanoparticles were resuspended in DI water. After the solution was repeatedly diluted 4 times, [email protected]
nanoparticle solution was mixed with 8-OHdG antibody (5 μL/mL) and incubated at 4 °C overnight. The mixture was purified by centrifugation (8000 rpm/min, 10 min) followed by DI water rinsing procedure. The biofunctionalized [email protected]
nanoparticles were blocked by BSA (1%). The mixture was centrifuged (8000 rpm/min, 10 min) and rinsed 3 times by DI water. The precipitate was dispersed in phosphate buffer saline (PBS) for later use.
Maximum excitation wavelength of synthetic CDs was determined by F2700 (Hitachi, Japan). Transmission electron microscopy (TEM) was applied to investigate the morphologies and sizes of [email protected]
nanoparticles and CDs by JEOL-2100F (JEOL, Japan). Powder X-ray diffraction was used to identify crystallography information by the X’Pert PRO X-ray diffractometer (PANalytical Ltd., Almelo, The Netherlands). Fourier-transform infrared (FTIR) spectra recorded chemical bonding information using Nicolet 6700 spectrometer (Thermo-Fisher, Waltham, MA, USA). The morphology of nanoporous alumina membranes were observed by SU8010 field emission scanning electron microscope equipped with an energy dispersive X-ray spectrometer attachment (SEM, Hitachi, Japan).
2.7. FRET Biosensor for 8-OHdG Detection
To investigate the detection of 8-OHdG by the FRET biosensor, various concentrations of 8-OHdG diluted in PBS (pH 7.4, 40 μL) were added to the functionalized nanoporous alumina membranes. They were reacted for 4 h at 37 °C. The membranes were rinsed to remove the unreacted chemicals. [email protected]
nanoparticles were modified by 8-OHdG antibody. The detection of 8-OHdG brought [email protected]
nanoparticles to CDs on nanoporous alumina membranes. The fluorescence intensity for various concentrations of 8-OHdG detection was recorded using F2700 ranging from 400 to 600 nm under 350 nm photoexcitation. The selectivity of the FRET biosensor was studied with the same concentration (3 μM) of NaCl, KCl, CaCl2
, thymine, cytosine, guanine, and adenine as interferences.
2.8. Detection of 8-OHdG in Urine Samples
Importantly, 8-OHdG was one kind of metabolites that appeared in urine. Urine samples were obtained from healthy young volunteers with consent. The protocol was approved by the ethics committee of Sir Run Run Shaw Hospital, an affiliate of Medical College, Zhejiang University (approval number: 20200831-34). Urine samples were collected and processed within an hour. They were centrifuged with the speed of 5000 rpm/min for 10 min. The supernatant was filtered by a 0.22 μm membrane diluted by PBS (pH = 7.4) with the dilution ratio of two times. The diluted supernatant (100 μL) was added to the FRET biosensor for 8-OHdG detection.