Polydopamine functionalized dendritic fibrous silica nanoparticles as a generic platform for nucleic acid-based biosensing

Accurate and rapid detection of nucleic acid sequences is of utmost importance in various fields, including disease monitoring, clinical treatment, gene analysis and drug discovery. In this study, we developed a "turn-on" fluorescence biosensor that enables simple and highly efficient detection of nucleic acid biomarkers. Our approach involves the utilization of 6-carboxyfluorescein modified single-stranded DNA (FAM-ssDNA) as molecular recognition element, along with polydopamine-functionalized dendritic fibrous nanosilica (DFNS). FAM-ssDNA serves as both specific molecular recognition element for the target analyte and reporter capable of transducing a detectable signal through Watson–Crick base pairing. The polydopamine-functionalized DFNS (DFNS@DA) exhibits strong binding to FAM-ssDNA via polyvalent metal mediated coordination leading to effective quenching by fluorescence resonance energy transfer. In the presence of a complementary target sequence, FAM-ssDNA forms hybridized structure and detaches from DFNS@DA, which causes an increased fluorescence emission. The analytical system based on FAM-ssDNA and DFNS@DA demonstrates exceptional sensitivity, selectivity, and rapid response for the detection of nucleic acid sequences, leveraging the high adsorption and quenching properties of DFNS@DA. For the first proof of concept, we demonstrated the successful detection of microRNA (miR-21) in cancer cells using the FAM-ssDNA/DFNS@DA system. Our results highlight the promising capabilities of DFNS@DA and nucleic acid-based biosensors, offering a generic and cost-effective solution for the detection of nucleic acid-related biomarkers. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00604-024-06234-2.


Experimental section
Preparation of SNS, DFNS-4 and DFNS-20 SNS were synthesized using a one-step Stöber procedure. 1A solution containing 100 mL of methanol, 33 mL of water, and 22.4 mL of ammonia (25%) was stirred using a magnetic stirrer in a 1000 mL glass beaker.A mixture of 130 mL of methanol containing 13.8 mL of TEOS was then rapidly added to the solution.The reaction mixture was stirred at room temperature for 8 h.The SNS were isolated by centrifugation, washed with water and methanol several times, and dried in a vacuum desiccator.DFNS-4 was synthesized by following a sol-gel reaction 2 , with slight modification.In a 100 mL round-bottom flask, 70 mL of water, 1 mL of aqueous ammonia (25%), 20 mL of ethyl ether, and 10 mL of ethanol were added and vigorously stirred with a magnetic stirrer at room temperature for 30 min, 500 mg of CTAB was then added to the mixture.After 30 min, a mixture of 2.5 mL of TEOS and 0.1 mL of APTES was added quickly into the abovementioned mixture.The reaction mixture was stirred vigorously at room temperature for 4 h.
Next, 1 mL of HCl (37%) was added to quench the reaction.The nanoparticles were isolated by centrifugation, washed with water and ethanol three times, and resuspended in 120 mL of ethanol by sonication, followed by addition of 15 mL of HCl (37%).The mixture was stirred at 70 °C for 24 h, and then the nanoparticles were isolated by centrifugation and washed with ethanol three times to remove the surfactant CTAB from DFNS.The DFNS-4 was collected by centrifugation and dried in a vacuum desiccator.DFNS-20 were synthesized following the procedure reported by Yu et al. 3 In a 250 mL round-bottomed flask, 100 mL of water and 0.272 g of TEA were added and stirred with a magnetic stirrer for 30 min at 80°C.Then, 1.52 g of CTAB and 0.672 g of NaSal were added to the mixture and stirred vigorously for 1 hour.After adding 16 mL of TEOS and 1 mL of ethanol, the reaction mixture was stirred vigorously for 2 h.The obtained nanoparticles were separated by centrifugation, washed with ethanol (three times), and then re-suspended in a mixture of HCl (37%) and methanol (1: 6 volume ratio) to remove CTAB.The particle suspension was stirred at 65 °C before the particles are separated and transferred into a new solution every 12 h.This process was repeated three times.Finally, the DFNS-20 were washed thoroughly with methanol and dried in a vacuum desiccator at room temperature overnight.

Fluorescence assays
The fluorescence spectra for probe were obtained at excitation and emission wavelengths of 488 and 518 nm, respectively.The following parameters were set for fluorescence measurement: a photomultiplier tube voltage of medium, a scan speed of medium, and excitation and emission slits of 10 nm.Fluorescence quenching efficiency (QE) was calculated using the formula QE = 1 − FM/F0, where FM and F0 represent the fluorescence intensities of probe in the presence and absence of NS@DA, respectively.Fluorescence recovery efficiency (RE) was calculated using the formula RE = FT/FM − 1, where FT and FM represent the fluorescence intensities of probe in the presence and absence of cDNA after NS@DA addition, respectively.
For the effect of the amounts of NS@DA, 2-16 μL of NS@DA solution was added to HEPES buffer (10 mM, pH 7.4) containing 2 mM CaCl2 (total volume 198 μL), then 2 μL of probe (20 µM) was added.After a 30 min incubation time at room temperature, the mixture was subjected to fluorescence measurements.For the effect of incubation temperature, 10 μL of SNS@DA solution was added to HEPES buffer (10 mM, pH 7.4) containing 2 mM CaCl2 (total volume 198 μL), then 2 μL of probe (20 µM) was added.After a 30 min incubation time at different temperatures, the mixture was subjected to fluorescence measurements.
For the kinetic studies of the fluorescence intensities of quenching and recovery, 10 μL of NS@DA solution was added to 188 μL HEPES buffer (10 mM, pH 7.4) containing 2 mM CaCl2, the mixture was transferred to a cuvette.After adding 2 μL of probe (20 µM), the fluorescence spectrum of the particle suspension was recorded every 0.5 min for 30 min.
After this step, 2 μL of cDNA (20 µM) was added to the cuvette, and the fluorescence spectrum was recorded every 0.5 min for 30 min.
For effect of metal ions for quenching, 10 μL of DFNS-20@DA solution was added to HEPES buffer (10 mM, pH 7.4) containing different concentration of CaCl2, MgCl2 and NaCl (total volume 198 μL), then 2 μL of probe (20 µM) was added.After a 30 min incubation time at room temperature, the mixture was subjected to fluorescence measurements.