A lateral flow strip for on-site detection of homocysteine based on a truncated aptamer.

An elevated level of homocysteine (Hcy) in serum is closely related to the development of various diseases. Therefore, homocysteine has been widely employed as a biomarker in medical diagnosis and the on-site detection of homocysteine is highly desired. In this study, a truncated highly specific aptamer for homocysteine was screened and used to design a lateral flow strip (LFS) for the detection of homocysteine. The aptamer was derived from a previously reported sequence. Based on the result of molecular docking, the original sequence was subjected to truncation, resulting in a reduction of the length from 66 nt to 55 nt. Based on the truncated aptamer, the LFS was designed for the detection of homocysteine. In the presence of homocysteine, the aptamer selectively binds to it, releasing cDNA from the aptamer/cDNA duplex. This allows cDNA to bind to the capture probe immobilized on the T zone of the strip, resulting in a red signal on the T zone from gold nanoparticles (AuNPs). The strip enables the visual detection of homocysteine in 5 min. Quantitative detection can be facilitated with the aid of ImageJ software. In this mode, the linear detection range for homocysteine is within 5-50 µM, with a detection limit of 4.18 µM. The strip has been effectively utilized for the detection of homocysteine in human serum. Consequently, the combination of the truncated aptamer and the strip offers a method that is sensitive, quick, and economical for the on-site detection of homocysteine.

A lateral flow assay for miRNA-21 based on CRISPR/Cas13a and MnO2 nanosheets-mediated recognition and signal amplification.

The point-of-care testing (POCT) of miRNA has significant application in medical diagnosis, yet presents challenges due to their characteristics of high homology, low abundance, and short length, which hinders the achievement of quick detection with high specificity and sensitivity. In this study, a lateral flow assay based on the CRISPR/Cas13a system and MnO2 nanozyme was developed for highly sensitive detection of microRNA-21 (miR-21). The CRISPR/Cas13a cleavage system exhibits the ability to recognize the specific oligonucleotide sequence, where two-base mismatches significantly impact the cleavage activity of the Cas13a. Upon binding of the target to crRNA, the cleavage activity of Cas13a is activated, resulting in the unlocking of the sequence and initiating strand displacement, thereby enabling signal amplification to produce a new sequence P1. When applying the reaction solution to the lateral flow test strip, P1 mediates the capture of MnO2 nanosheets (MnO2 NSs) on the T zone, which catalyzes the oxidation of the pre-immobilized colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) on the T zone and generates the blue-green product (ox-TMB). The change in gray value is directly proportional to the concentration of miR-21, allowing for qualitative detection through visual inspection and quantitative measurement using ImageJ software. This method achieves the detection of miR-21 within a rapid 10-min timeframe, and the limit of detection (LOD) is 0.33 pM. With the advantages of high specificity, simplicity, and sensitivity, the lateral flow test strip and the design strategy hold great potential for the early diagnosis of related diseases.

pH-Responsive Pesticide-Loaded Hollow Mesoporous Silica Nanoparticles with ZnO Quantum Dots as a Gatekeeper for Control of Rice Blast Disease.

Nanotechnology-enabled pesticide delivery systems have been widely studied and show great prospects in modern agriculture. Nanodelivery systems not only achieve the controlled release of agrochemicals but also possess many unique characteristics. This study presents the development of a pH-responsive pesticide nanoformulation utilizing hollow mesoporous silica nanoparticles (HMSNs) as a nanocarrier. The nanocarrier was loaded with the photosensitive pesticide prochloraz (Pro) and then combined with ZnO quantum dots (ZnO QDs) through electrostatic interactions. ZnO QDs serve as both the pH-responsive gatekeeper and the enhancer of the pesticide. The results demonstrate that the prepared nanopesticide exhibits high loading efficiency (24.96%) for Pro. Compared with Pro technical, the degradation rate of Pro loaded in HMSNs@Pro@ZnO QDs was reduced by 26.4% after 24 h ultraviolet (UV) exposure, indicating clearly improved photostability. In a weak acidic environment (pH 5.0), the accumulated release of the nanopesticide after 48 h was 2.67-fold higher than that in a neutral environment. This indicates the excellent pH-responsive characteristic of the nanopesticide. The tracking experiments revealed that HMSNs can be absorbed by rice leaves and subsequently transported to other tissues, indicating their potential for effective systemic distribution and targeted delivery. Furthermore, the bioactivity assays confirmed the fungicidal efficacy of the nanopesticide against rice blast disease. Therefore, the constructed nanopesticide holds great prospect in nanoenabled agriculture, offering a novel strategy to enhance pesticide utilization.

Study of dual binding specificity of aptamer to ochratoxin A and norfloxacin and the development of fluorescent aptasensor in milk detection.

Target specificity, one of aptamer characteristics that determine recognition efficiency of biosensors, is generally considered to be an intrinsic property of aptamer. However, a high-affinity aptamer may have additional target binding specificity, little is known about the specificity of aptamer binding to multiple targets, which may result in false-positive results that hinder the accuracy of detection. Herein, an aptamer OBA3 with dual target ochratoxin A (OTA) and norfloxacin (NOR) was used as an example to explore the binding specificity mechanism and developed rapid fluorescent aptasensing methods. The nucleotide 15th T of aptamer OBA3 was demonstrated to be critical for specificity and affinity binding of target OTA via site-saturation mutagenesis. Substituting the 15th T base for C base could directly improve recognition specificity of aptamer for NOR and remove the binding affinity for OTA. The combination of π-π stacking interactions, salt bridges and hydrogen bonds between loop pocket of aptamer and quinolone skeleton, piperazinyl group may contributes to the fluoroquinolone antibiotics (NOR and difloxacin)-aptamer recognition interaction. Based on this understanding, a dual-aptamer fluorescent biosensor was fabricated for simultaneous detection of OTA and NOR, which has a linear detection range of 50-6000 nM with a detection limit of 31 nM for OTA and NOR. Combined with T15C biosensor for eliminating interference of OTA, the assay was applied to milk samples with satisfactory recovery (94.06-100.93%), which can achieve detection of OTA and NOR individually within 40 min.

A FRET based ultrasensitive fluorescent aptasensor for 6'-sialyllactose detection.

As a kind of human milk oligosaccharide, 6'-sialyllactose (6'-SL) plays an important role in promoting infant brain development and improving infant immunity. The content of 6'-SL in infant formula milk powder is thus one of the important nutritional indexes. Since the lacking of efficient and rapid detection methods for 6'-SL, it is of great significance to develop specific recognition elements and establish fast and sensitive detection methods for 6'-SL. Herein, using 6'-SL specific aptamer as the recognition element, catalytic hairpin assembly as the signal amplification technology and quantum dots as the signal label, a fluorescence biosensor based on fluorescence resonance energy transfer (FRET) was constructed for ultra-sensitive detection of 6'-SL. The detection limit of this FRET-based fluorescent biosensor is 0.3 nM, and it has some outstanding characteristics such as high signal-to-noise ratio, low time-consuming, simplicity and high efficiency in the actual sample detection. Therefore, it has broad application prospect in 6'-SL detection.

Ratiometric fluorescent biosensor for detection and real-time imaging of nitric oxide in mitochondria of living cells.

Nitric oxide (NO), a ubiquitous gaseous messenger, plays critical roles in various pathological and physiological progresses. The abnormal levels of NO in organisms are closely related to a large number of maladies. Mitochondria are the main area that produce NO in mammalian cells. Thus, detecting and real-time imaging of NO in mitochondria is of great significance for exploring the biological functions of NO. Herein, a ratiometric fluorescent biosensor (Mito-GNP-pNO520) is developed for sensitive and selective detection and real-time imaging of NO in mitochondria of living cells. The detection is achieved through the fluorescence off-on response of Mito-GNP-pNO520 toward NO. This biosensor shows excellent characteristics, such as high sensitivity toward NO with a low detection limit of 0.25 nM, exclusive selectivity to NO without interference from other substances, good biological stability and low cytotoxicity. More importantly, the biosensor is specifically located in mitochondria, enabling the detection and real-time imaging of endogenous and exogenous NO in mitochondria of living cells. Therefore, our biosensor offers a new approach for dynamic detecting and real-time imaging of NO in subcellular organelles, providing an opportunity to explore new biological effects of NO.

Graphene oxide-enhanced colorimetric detection of Mec A gene based on toehold-mediated strand displacement.

Mec A, as a representative gene mediating resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA), allows a new genetic analysis for the detection of MRSA. Here, a sensitive, prompt, and visual colorimetry is reported to detect the Mec A gene based on toehold-mediated strand displacement (TMSD) and the enrichment effect of graphene oxide (GO). The Mec A triggers to generate the profuse amount of signal units of single-stranded DNA (SG) composed of a long single-stranded base tail and a base head: the tail can be adsorbed and enriched on the surface of GO; the head can form a G quadruplex structure to exert catalytic function towards 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Therefore, through the enrichment effect of GO, the signal units SG reflects different degrees of signal amplification on different substrates (such as aqueous solution or filter membrane). This strategy demonstrates a broad linear working range from 100 pM to 1.5 nM (solution) and 1 pM to 1 nM (filter membrane), with a low detection limit of 39.53 pM (solution) and 333 fM (filter membrane). Analytical performance in real samples suggests that this developed colorimetry is endowed with immense potential for clinical detection applications.

Enhanced sensitivity in Staphylococcus aureus detection: Unveiling the impact of lipid composition on the performance of carboxyfluorescein (CF)-Loaded liposome-based assay.

Liposomes have emerged as versatile nanocarriers, finding applications not only in drug delivery but also in pathogen detection and diagnostics. This study aimed to enhance the sensitivity of liposomes to Staphylococcus aureus by investigating the impact of lipid composition on liposomes loaded with 5(6)-carboxyfluorescein (CF). Liposomes were fabricated using various concentrations of cholesterol (10-40 mol%) combined with saturated phospholipids. Dynamic light scattering results revealed that higher cholesterol concentrations led to reduced liposome size, CF release (%), and entrapment efficiency (%). Liposome sensitivity towards S. aureus was evaluated by using CF-loaded liposomes with and without aptamer insertion. Liposomes with a higher cholesterol content (40 mol%) exhibited a strong ability to detect low bacterial concentrations down to 5 × 102 CFU/mL without relying solely on specific receptor-ligand recognition. However, functionalizing the liposome with an aptamer further improved the specificity and sensitivity of S. aureus detection at even lower concentrations, down to 80 CFU/mL, in the wide range of 80-107 CFU/mL. This study highlights the potential for optimizing the lipid composition of liposomes to improve their sensitivity for pathogen detection, particularly when combined with aptamer-based strategies.

Nanosensor Based on the Dual-Entropy-Driven Modulation Strategy for Intracellular Detection of MicroRNA.

The entropy-driven strategy has been proposed as a milestone work in the development of nucleic acid amplification technology. With the characteristics of an enzyme-free, isothermal, and relatively simple design, it has been widely used in the field of biological analysis. However, it is still a challenge to apply entropy-driven amplification for intracellular target analysis. In this study, a dual-entropy-driven amplification system constructed on the surface of gold nanoparticles (AuNPs) is developed to achieve fluorescence determination and intracellular imaging of microRNA-21 (miRNA-21). The dual-entropy-driven amplification strategy internalizes the fuel chain to avoid the complexity of the extra addition in the traditional entropy-driven amplification strategy. The unique self-locked fuel chain system is established by attaching the three-stranded structure on two groups of AuNPs, where the Cy5 fluorescent label was first quenched by AuNPs. After the target miRNA-21 is identified, the fuel chain will be automatically unlocked, and the cycle reaction will be driven, leading to fluorescence recovery. The self-powered and waste-recycled fuel chain greatly improves the automation and intelligence of the reaction process. Under the optimal conditions, the linear response range of the nanosensor ranges from 5 pM to 25 nM. This nanoreaction system can be used to realize intracellular imaging of miRNA-21, and its good specificity enables it to distinguish tumor cells from healthy cells. The development of the dual-entropy-driven strategy provides an integrated and powerful way for intracellular miRNA analysis and shows great potential in the biomedical field.

Systematic bio-fabrication of aptamers and their applications in engineering biology.

Aptamers are single-stranded DNA or RNA molecules that have high affinity and selectivity to bind to specific targets. Compared to antibodies, aptamers are easy to in vitro synthesize with low cost, and exhibit excellent thermal stability and programmability. With these features, aptamers have been widely used in biology and medicine-related fields. In the meantime, a variety of systematic evolution of ligands by exponential enrichment (SELEX) technologies have been developed to screen aptamers for various targets. According to the characteristics of targets, customizing appropriate SELEX technology and post-SELEX optimization helps to obtain ideal aptamers with high affinity and specificity. In this review, we first summarize the latest research on the systematic bio-fabrication of aptamers, including various SELEX technologies, post-SELEX optimization, and aptamer modification technology. These procedures not only help to gain the aptamer sequences but also provide insights into the relationship between structure and function of the aptamers. The latter provides a new perspective for the systems bio-fabrication of aptamers. Furthermore, on this basis, we review the applications of aptamers, particularly in the fields of engineering biology, including industrial biotechnology, medical and health engineering, and environmental and food safety monitoring. And the encountered challenges and prospects are discussed, providing an outlook for the future development of aptamers.

Quantitative Determination of Staphylococcus aureus Using Aptamer-Based Recognition and DNA Amplification Machinery.

Staphylococcus aureus (S. aureus) is a common foodborne pathogen that threatens human health and safety. It is significant to develop sensitive detection methods for the monitoring of S. aureus contamination in food and environment. Herein, a novel machinery based on aptamer recognition, DNA walker, and rolling circle amplification (RCA) was designed, which can form unique DNA nanoflower and subsequently detect low-level S. aureus contamination in samples. To this end, two rationally designed DNA duplexes were modified on the surface of the electrode to identify S. aureus through the high affinity between aptamers and S. aureus. Combined with the repeated movement of DNA walker machinery on the electrode surface and RCA technology, a unique DNA nanoflower structure was formed. This can effectively transform the biological information of aptamer recognition of S. aureus into a significantly amplified electrochemical signal. Through reasonable design and optimization of the parameters of each part, the linear response range of the S. aureus biosensor is from 60 to 6 × 107 CFU/mL and the detection limit is as low as 9 CFU/mL.

Immobilization-free electrochemical homogeneous aptasensor for highly sensitive detection of carcinoembryonic antigen by dual amplification strategy.

Electrochemical aptasensor has been widely studied, while its practical application is limited by the unavoidable variations of aptamer loading densities and low signal amplification efficiency. To overcome these restrictions, an immobilization-free and label-free electrochemical homogeneous aptasensor was constructed for carcinoembryonic antigen (CEA) assay by combining RecJf exonuclease-mediated target cycling strategy and rolling circle amplification technology. In this system, the pre-immobilization of aptamers or other relevant signal elements on the electrode substrate is no longer necessary, thus the electrochemical homogeneous aptasensor shows good versatility on different transducers. Moreover, the whole recognition and signal amplification process are activated instantaneously by a non-professional operation of the solution mixture. This strategy can not only increase the stability (95.1% after 30 days of storage) and reproducibility (2.12% among five independent electrodes), but also further improve the sensitivity (detection limit of fg mL-1 level) due to the free target recognition and dual signal amplification in the homogeneous solution phase. The proposed immobilization-free electrochemical homogeneous aptasensors on different electrode substrates both achieve satisfactory results in actual sample tests, which has the potential for commercial applications and the establishment of other target platforms in the future.

Development of a Fluorescent Biosensor Based on DNAzyme for Tracing the Release of Zinc in Maize Leaves.

A fluorescent biosensor for real-time monitoring the release of Zn2+ in plants was constructed through immobilization of DNAzyme-containing hairpin DNA on nanofertilizer ZnO@Au nanoparticles (ZnO@Au NPs). A specially designed hairpin DNA containing both DNAzyme and its substrate sequence, which was also labeled with 5'-FAM and 3'-SH groups, was modified on ZnO@Au NPs through the Au-S bond. The fluorescent signal of FAM was initially quenched by AuNPs. When Zn2+ was released from ZnO@Au NPs, DNAzyme was activated and the substrate sequence in hairpin DNA was cleaved. The restored fluorescent signal in Tris-HCl buffer (pH 6.5) was correlated with the concentration of the released Zn2+. The performance of the biosensor was first demonstrated in the solution. The linear detection range was from 50 nM to 1.5 µM, with a detection limit of 30 nM. The biosensor system can penetrate into maize leaves with ZnO@Au NPs. With the release of Zn2+ in leaves, the restored fluorescence can be imaged by a confocal laser scanning microscope and used for monitoring the release and distribution of Zn2+. This work may provide a novel strategy for tracing and understanding the mechanism of nanofertilizers in organisms.

Correction: A fluorescent aptasensor for ATP based on functional DNAzyme/walker and terminal deoxynucleotidyl transferase-assisted formation of DNA-AgNCs.

Correction for 'A fluorescent aptasensor for ATP based on functional DNAzyme/walker and terminal deoxynucleotidyl transferase-assisted formation of DNA-AgNCs' by Shixin Cai et al., Analyst, 2023, 148, 799-805, https://doi.org/10.1039/D2AN02006H.

A fluorescent aptasensor for ATP based on functional DNAzyme/walker and terminal deoxynucleotidyl transferase-assisted formation of DNA-AgNCs.

The development of sensitive adenosine triphosphate (ATP) sensors is imperative due to the tight relationship between the physiological conditions and ATP levels in vivo. Herein, a fluorescent aptasensor for ATP is presented, which adopts a strategy that combines a split aptamer and a DNAzyme/walker with terminal deoxynucleotidyl transferase (TDT)-assisted formation of DNA-AgNCs to realize fluorescence detection of ATP. A multifunctional oligonucleotide sequence is rationally designed, which integrates a split aptamer, a DNAzyme and a DNA walker. Both multifunctional oligonucleotide and its substrate strand are connected to the surface of Fe3O4@Au nanoparticles via Au-S bonds. The existence of ATP can induce the formation of the complete aptamer, and then activate the DNAzyme to circularly cleave the substrate strand, leaving 2',3'-cyclophosphate at the 3'end of the strand. This blocks the polymerization of dCTP to form poly(C) even in the presence of TDT and dCTP, due to the lack of free 3'-OH. In contrast, when ATP is absent, the DNAzyme/walker cannot work and then TDT catalyzes the formation of poly(C) at the free 3'-OH of the substrate strand, which is subsequently utilized as the template to prepare DNA-AgNCs. The fluorescence response derived from AgNCs thus reflects the ATP concentration. Under the optimum conditions, the aptasensor shows a linear response range from 5 nM to 10 000 nM, with a detection limit of 0.27 nM. The level of ATP in human serum can be effectively measured by the aptasensor with good recovery, indicating its application potential in medical samples.

Screening of a Sialyllactose-Specific Aptamer and Engineering a Pair of Recognition Elements with Unique Fluorescent Characteristics for Sensitive Detection of Sialyllactose.

A single-stranded DNA (ssDNA) aptamer specific for 6'-sialyllactose (6'-SL) was screened through magnetic separation-based SELEX and post-SELEX truncation and used to construct unique aptamer bio-dots for sensitive detection of 6'-SL. Eighteen rounds of screening were conducted during the SELEX process. The ssDNA aptamer Apt9 (Kd = 152.3 nM) with a length of 79 nucleotides (nt) was demonstrated as the optimal aptamer candidate after affinity and specificity evaluation. Then, Apt9 was truncated and optimized according to secondary structure and molecular docking. A 35 nt truncated aptamer Apt9-1 (Kd = 91.75 nM) with higher affinity than Apt9 was finally obtained. Furthermore, Apt9-1 was used to synthesize bio-dots as a new recognition element of 6'-SL, and the aminobenzene boric acid functionalized carbon dots were employed as the other recognition element. With the respective fluorescent characteristics, the two quantum dots (QDs) were made a pair to construct a 6'-SL fluorescent biosensor. The linear detection range of the biosensor is 10 µM to 5 mM, and the detection limit is 0.9 µM. With the advantages of time-saving, high efficiency, and simplicity in the actual sample detection, the screened aptamer and dual-QD-based biosensor have broad application prospects in 6'-SL detection.

Bilayer magnetic-plasmonic satellite nanoassemblies for SERS detection of tobramycin with exonuclease amplification.

Nanoscale assemblies designed for trace analyte detection typically require a complex fabrication process. Here, we prepare magnetic nanoparticle (Fe3O4)-gold nanoparticle (AuNP)-gold nanostar (AuNS) bilayer magnetic-plasmonic satellite nanoassemblies (BMPSNs) for ultrasensitive detection of tobramycin (TOB). BMPSNs are constructed through seed-mediated growth and complementary DNA hybridization, combining magnetic separation and surface-enhanced Raman scattering (SERS) activities. AuNP is in situ growth on the surface of Fe3O4 to form the monolayer satellite assemblies. Partially complementary double-stranded DNA (DNA1/DNA2) is modified onto the surface of the first layer satellite AuNP. TOB aptamer (Apt) and fully complementary DNA (cDNA) form the duplex DNA. In the presence of TOB, cDNA of TOB Apt is replaced by TOB/TOB Apt, which can hybridize with DNA2 modified on the surface of Fe3O4@AuNP-DNA1/DNA2 and further triggers exonuclease III cyclic amplification to obtain Fe3O4@AuNP-DNA1. Finally, Fe3O4@AuNP-DNA1 can assemble with AuNS@4-MBA-DNA3 through DNA hybridization to form BMPSNs. Thanks to excellent magnetic separation, exonuclease amplification and huge SERS enhancement of multiple hot spots, the limit of detection can achieve as low as 0.44 fg/mL of TOB, which is more sensitive than the previously reported methods. In addition, this method can be applied to TOB detection in actual samples with good recoveries and without interference by other antibiotics. The proposed method can be easily extended to sensitive detection of other targets by replacing the corresponding aptamers, paving a new avenue for food safety and environment monitoring.

A surface-enhanced Raman scattering aptasensor for Escherichia coli detection based on high-performance 3D substrate and hot spot effect.

A surface-enhanced Raman scattering (SERS) aptasensor was established for highly sensitive and selective detection of Escherichia coli (E. coli). Chitosan hydrogel modified with E. coli aptamer (Apt) functionalized silver nanoparticles was constructed as a SERS 3D substrate for specific bacteria enrichment, while the Raman signaling molecule 4-mercaptobenzoic acid and E. coli Apt modified gold nanostars were prepared for the sensitive quantification of E. coli. The aptasensor exhibits intense electromagnetic field enhancement in multiple hot spot regions, including the spikes and the gap between adjacent nanostars and that between gold nanostars and silver nanoparticles. Due to the hot spot effect coupled with the selective recognition ability, a detection limit of 3.46 CFU/mL with a wide dynamic linearized range from 3.2 × 101 to 3.2 × 107 CFU/mL could be achieved without other non-target bacteria interference. Moreover, this SERS aptasensor was applied to detect E. coli in actual samples with a good recovery rate (>90%). Therefore, the developed SERS aptasensor paves a new avenue for the detection in the field of food safety and environmental pollution by replacing the corresponding aptamers.

An ultrasensitive and dual-recognition SERS biosensor based on Fe3O4@Au-Teicoplanin and aptamer functionalized Au@Ag nanoparticles for detection of Staphylococcus aureus.

An ultrasensitive and dual-recognition surface-enhanced Raman scattering (SERS) biosensor for Staphylococcus aureus (S. aureus) was constructed, which was based on teicoplanin (Tcp) functionalized gold-coated magnet nanoparticles (Fe3O4@Au-Tcp NPs) as capture probe and S. aureus aptamer (Apt) functionalized silver coated gold nanoparticles (Au@Ag-DTNB-Apt NPs) as signal probe. Both Au NPs and Au@Ag NPs were prepared by a green synthesis method. Especially, the synthesis method of Au@Ag NPs reduced by chitosan (CS) was first reported in this work. Due to the great SERS enhancement based on the hot spot effect between Au NPs and Au@Ag NPs, and the dual-recognition ability based on Tcp and Apt, the SERS biosensor was ultrasensitive and specific. A detection limit of 1.09 CFU mL-1 with a broad dynamic linear (7.6 × 101-7.6 × 107 CFU mL-1) was achieved within 50 min without interference by other bacteria. Moreover, the SERS biosensor could be applied for detection of S. aureus in milk and orange juice samples. This study provides a green, rapid and ultrasensitive method to detect S. aureus, and also explores the high utilization value of CS and Tcp, which has a broad application prospect in detection of pathogenic bacteria.

A pH-Gated Functionalized Hollow Mesoporous Silica Delivery System for Photodynamic Sterilization in Staphylococcus aureus Biofilm.

Multidrug-resistant bacteria are increasing, particularly those embedded in microbial biofilm. These bacteria account for most microbial infections in humans. Traditional antibiotic treatment has low efficiency in sterilization of biofilm-associated pathogens, and thus the development of new approaches is highly desired. In this study, amino-modified hollow mesoporous silica nanoparticles (AHMSN) were synthesized and used as the carrier to load natural photosensitizer curcumin (Cur). Then glutaraldehyde (GA) and polyethyleneimine (PEI) were used to seal the porous structure of AHMSN by the Schiff base reaction, forming positively charged AHMSN@GA@PEI@Cur. The Cur delivery system can smoothly diffuse into the negatively charged biofilm of Staphylococcus aureus (S. aureus). Then Cur can be released to the biofilm after the pH-gated cleavage of the Schiff base bond in the slightly acidic environment of the biofilm. After the release of the photosensitizer, the biofilm was irradiated by the blue LED light at a wavelength of 450 nm and a power of 37.4 mV/cm2 for 5 min. Compared with the control group, the number of viable bacteria in the biofilm was reduced by 98.20%. Therefore, the constructed pH-gated photosensitizer delivery system can efficiently target biofilm-associated pathogens and be used for photodynamic sterilization, without the production of antibiotic resistance.