Nanoparticle Probes Research Articles

CRISPR-Cas12a-based nanoparticle biosensor for detection of pathogenic bacteria in food

Pathogenic bacterial contamination of food is a major challenge in food safety and public health management. To monitor pathogenic bacteria in food, a rapid, sensitive, and specific pathogen detection method is needed. CRISPR-Cas12a has been developed as a sensitive method for detecting pathogenic bacteria, but its output signal is fluorescent, which need equipment to detect. Here, the nanoparticle probe was designed to detect the activity of Cas12a and were able to visualize the assay results by observing the color change by naked eyes. The target sequences of pathogenic bacteria were amplified by recombinase polymerase, then recognized and bound by the crRNA and Cas12a complex. The activated Cas12a cleaved the linker-ssDNA connecting the nanoparticles, causing the nanoparticles to transition from aggregation to dispersion, and the color showed as purple. The sensitivity analysis showed that the limits of detection (LOD) reached 1 × 10°CFU/mL, 1 × 104 CFU/mL, 1 × 102 CFU/mL, 1 × 103 CFU/mL, 1 × 101 CFU/mL for Escherichia coli, Salmonella typhimurium, Vibrio parahaemolyticus, Staphylococcus aureus, and Listeria monocytogenes, which were all specific for five bacteria in the milk samples. Therefore, this method is a dependable, fast, sensitive and specific method, which make the CRIPSR-Cas12a detection method more useful in bacterial pathogenic detection.

A dual-mode colorimetric and surface-enhanced Raman strategy for chloramphenicol detection based on the Au@Pt nanozyme and EXPAR

The misuse of chloramphenicol (CAP) has resulted in an escalation of antibiotic-resistant bacterial strains, posing a significant threat to human health. Through the fabrication of magnetic multi-“hotspot” nanoflower particles as SERS substrates and combine bimetallic peroxidase Au@Pt, dual-mode detection of CAP was achieved. The preparation of magnetic nanoparticles Fe3O4@Au facilitated rapid aggregation, enriching a SERS signal. Integrated with the exponential amplification reaction (EXPAR) strategy, under isothermal conditions, a highly rate increase of amplicon production was achieved, thereby binding more nanozyme Au@Pt. In the presence of the target CAP, due to specific binding with the aptamer, the EXPAR was activated, generating a substantial amount of amplicons that formed a stable “Y-shaped” structure with magnetic nanoparticle probes and nanozyme probes. With the addition of TMB, the excellent peroxidase-like activity of the nanozyme oxidized it to oxTMB, resulting in changes in both color and Raman signals. This strategy achieved dual-mode ultra-trace detection of CAP. The SERS detection ranged from 1.0 × 10-12 M to 1.0 × 10-6 M, with a detection limit of 4.96 × 10-13 M; the colorimetric method ranged from 2.5 × 10-7 M to 1.0 × 10-8 M, with a detection limit of 9.23 × 10-9 M. This method offers a new approach and insights for the ultra-trace detection of antibiotics.

A label-free and signal-amplifiable fluorescent biosensor based on aptamer-conjugated gold nanoparticles and hybridization chain reaction for determination of carcinoembryonic antigen.

The sensitive detection of cancer biomarkers is crucial for early accurate diagnostics and therapy of cancer patients. Carcinoembryonic antigen (CEA) is a tumor-associated antigen derived from colon cancer and embryonic tissues. In this study, we have developed a label-free fluorescence biosensing platform for the quantification of CEA with the "turn-on" signal output. This platform employs a label-free strategy that incorporates an aptamer-modified gold nanoparticle (Apt@AuNP) probe for the recognition of CEA, in combination with hybridization chain reaction (HCR) amplification. In the presence of target CEA, Apt@AuNPs selectively capture CEA, resulting in a reduction of subsequent complementary chains (CP) binding on Apt@AuNPs. The remaining CP, acting as the initiator sequence for HCR, triggers the HCR, leading to the formation of abundant G-quadruplex structures. By employing Thioflavin T (ThT) for the formation of G-quadruplex/ThT complexes, the biosensor exhibits a significant enhancement of the fluorescence signal. Under optimized conditions, the biosensor platform demonstrates a limit of detection of 0.03 nM and a linear range from 0.1 to 2.5nM. Additionally, the specificity investigation reveals the high selectivity of this fluorescent biosensor. Finally, the performance of this method has been validated by successfully detecting CEA in real-life samples, highlighting its potential for clinical applications.

The Current Status and Future Directions on Nanoparticles for Tumor Molecular Imaging.

Molecular imaging is an advanced technology that utilizes specific probes or markers in conjunction with cutting-edge imaging techniques to observe and analyze the localization, distribution, activity, and interactions of biomolecules within living organisms. Tumor molecular imaging, by enabling the visualization and quantification of molecular characteristics of tumor cells, facilitates a deeper and more comprehensive understanding of tumors, providing valuable insights for early diagnosis, treatment monitoring, and cancer biology research. However, the image quality of molecular imaging still requires improvement, and nanotechnology has significantly propelled the advancement of molecular imaging. Currently, nanoparticle-based tumor molecular imaging technologies encompass radionuclide imaging, fluorescence imaging, magnetic resonance imaging, ultrasound imaging, photoacoustic imaging, and multimodal imaging, among others. As our understanding of the tumor microenvironment deepens, the design of nanoparticle probes for tumor molecular imaging has also evolved, offering new perspectives and expanding the applications of tumor molecular imaging. Beyond diagnostics, there is a marked trend towards integrated diagnosis and therapy, with image-guided treatment playing a pivotal role. This includes image-guided surgery, photodynamic therapy, and chemodynamic therapy. Despite continuous advancements and innovative developments in molecular imaging, many of these remain in the experimental stage and require breakthroughs before they can be fully integrated into clinical practice.

Visualizing Single V-ATPase Rotation Using Janus Nanoparticles.

Understanding the function of rotary molecular motors, such as the rotary ATPases, relies on our ability to visualize the single-molecule rotation. Traditional imaging methods often involve tagging those motors with nanoparticles (NPs) and inferring their rotation from translational motion of NPs. Here, we report an approach using "two-faced" Janus NPs to directly image the rotation of single V-ATPase from Enterococcus hirae, an ATP-driven rotary ion pump. By employing a 500-nm silica/gold Janus NP, we exploit its asymmetric optical contrast - silica core with a gold cap on one hemisphere - to achieve precise imaging of the unidirectional counter-clockwise rotation of single V-ATPase motors immobilized on surfaces. Despite the added viscous load from the relatively large Janus NP probe, our approach provides accurate torque measurements of single V-ATPase. This study underscores the advantages of Janus NPs over conventional probes, establishing them as powerful tools for single-molecule analysis of rotary molecular motors.

Detection of Tumor-Associated Autoantibodies in the Sera of Pancreatic Cancer Patients Using Engineered MUC1 Glycopeptide Nanoparticle Probes.

Pancreatic cancer is one of the deadliest cancers worldwide, mainly due to late diagnosis. Therefore, there is an urgent need for novel diagnostic approaches to identify the disease as early as possible. We have developed a diagnostic assay for pancreatic cancer based on the detection of naturally occurring tumor associated autoantibodies against Mucin-1 (MUC1) using engineered glycopeptides on nanoparticle probes. We used a structure-guided approach to develop unnatural glycopeptides as model antigens for tumor-associated MUC1. We designed a collection of 13 glycopeptides to bind either SM3 or 5E5, two monoclonal antibodies with distinct epitopes known to recognize tumor associated MUC1. Glycopeptide binding to SM3 or 5E5 was confirmed by surface plasmon resonance and rationalized by molecular dynamics simulations. These model antigens were conjugated to gold nanoparticles and used in a dot-blot assay to detect autoantibodies in serum samples from pancreatic cancer patients and healthy volunteers. Nanoparticle probes with glycopeptides displaying the SM3 epitope did not have diagnostic potential. Instead, nanoparticle probes displaying glycopeptides with high affinity for 5E5 could discriminate between cancer patients and healthy controls. Remarkably, the best-discriminating probes show significantly better true and false positive rates than the current clinical biomarkers CA19-9 and carcinoembryonic antigen (CEA).

Detection of Tumor‐Associated Autoantibodies in the Sera of Pancreatic Cancer Patients Using Engineered MUC1 Glycopeptide Nanoparticle Probes

AbstractPancreatic cancer is one of the deadliest cancers worldwide, mainly due to late diagnosis. Therefore, there is an urgent need for novel diagnostic approaches to identify the disease as early as possible. We have developed a diagnostic assay for pancreatic cancer based on the detection of naturally occurring tumor associated autoantibodies against Mucin‐1 (MUC1) using engineered glycopeptides on nanoparticle probes. We used a structure‐guided approach to develop unnatural glycopeptides as model antigens for tumor‐associated MUC1. We designed a collection of 13 glycopeptides to bind either SM3 or 5E5, two monoclonal antibodies with distinct epitopes known to recognize tumor associated MUC1. Glycopeptide binding to SM3 or 5E5 was confirmed by surface plasmon resonance and rationalized by molecular dynamics simulations. These model antigens were conjugated to gold nanoparticles and used in a dot‐blot assay to detect autoantibodies in serum samples from pancreatic cancer patients and healthy volunteers. Nanoparticle probes with glycopeptides displaying the SM3 epitope did not have diagnostic potential. Instead, nanoparticle probes displaying glycopeptides with high affinity for 5E5 could discriminate between cancer patients and healthy controls. Remarkably, the best‐discriminating probes show significantly better true and false positive rates than the current clinical biomarkers CA19‐9 and carcinoembryonic antigen (CEA).

A Lateral Flow Biosensor Based on Isothermal Amplification for Visual Identification of Species and Sex from Bloodstain.

The prompt species identification from biological samples at a crime scene can rapidly filter out truly valuable biometric information for subsequent personal identification. Meanwhile, early sex determination can assist in narrowing the pool of suspects. However, the current methods for forensic DNA analysis, particularly in point-of-care scenarios, are often limited by the intricate equipment for signal generation and the laborious procedure for DNA purification. The present study introduces a novel portable lateral flow biosensor that possesses extraction-free and anti-aerosol characteristics for on-site determination of species and sex. The bloodstain can be directly submitted to loop-mediated isothermal amplification (LAMP) for the analysis of both mitochondrial and nuclear DNA. The incorporation of a lateral flow device with gold magnetic nanoparticle probes allows for visual interpretation of results through colorimetric signals while also preventing interference on result judgment from pigments such as hemoglobin. Carryover contamination, which is a disharmonious factor in LAMP, especially as the inherent contradiction derived from uncapping in the lateral flow strategy, has been effectively addressed through the integration of uracil DNA glycosylase without compromising the isothermy throughout the process. As a proof-of-concept experiment, species and sex can be accurately identified within 40 min from trace bloodstains amidst significant background interference by targeting cytochrome b and Y-chromosomal amelogenin. Furthermore, the single-blind study revealed a concordance rate of up to 100% in both simulative degraded and true dated bloodstains. This suggests that this biosensor has the potential to be utilized in forensic DNA analysis at crime scenes.

Rapid cyprinid herpesvirus 3 detection using loop-mediated isothermal amplification (LAMP) combined with gold nanoprobes and SYBR safe

As an etiological agent of a notifiable and emerging disease, Cyprinid herpesvirus 3 (CyHV-3 or koi herpesvirus, KHV) is a highly risky factor that affects koi and common carp yield through increasing the mortality rates. In the current study, a highly rapid and sensitive technique was established to detect CyHV-3 with the help of loop-mediated isothermal amplification (LAMP) method with dual visualizing indicators (SYBR safe and gold nanoparticle probes; AuNP-probes). Six specific primers were used to amplify the thymidine kinase (TK) of CyHV-3 using a LAMP reaction of 45 min at 65 °C along with detecting the products through visual inspection via hybridization at 65 °C for 20 min with a functionalized thiol-AuNP probe. A closed-tube LAMP-SYBR safe assay was also developed under the same condition without AuNP probe. The amplification of KHV-LAMP products were evaluated by the naked eye, through fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric aggregate of LAMP product in the presence of AuNP probe. Furthermore, quantitative measurement was applied to the LAMP-AuNPs assay, using the spectral shift assay. The detection limit was 1.25 fentograms DNA which can compete the latest techniques available. The proposed technique yielded negative outcomes with DNA template from other viruses such as CyHV-1 and CEV. The LAMP-SYBR safe and LAMP-AuNPs assays introduced in this article were very sensitive, fast, and reliable diagnostic tools for the detection of CyHV-3.

Dual-Mode Fluorescent/Intelligent Lateral Flow Immunoassay Based on Machine Learning Algorithm for Ultrasensitive Analysis of Chloroacetamide Herbicides.

Given the harmful effect of pesticide residues, it is essential to develop portable and accurate biosensors for the analysis of pesticides in agricultural products. In this paper, we demonstrated a dual-mode fluorescent/intelligent (DM-f/DM-i) lateral flow immunoassay (LFIA) for chloroacetamide herbicides, which utilized horseradish peroxidase-IgG conjugated time-resolved fluorescent nanoparticle probes as both a signal label and amplification tool. With the newly developed LFIA in the DM-f mode, the limits of detection (LODs) were 0.08 ng/mL of acetochlor, 0.29 ng/mL of metolachlor, 0.51 ng/mL of Propisochlor, and 0.13 ng/mL of their mixture. In the DM-i mode, machine learning (ML) algorithms were used for image segmentation, feature extraction, and correlation analysis to obtain multivariate fitted equations, which had high reliability in the regression model with R2 of 0.95 in the range of 2 × 102-2 × 105 pg/mL. Importantly, the practical applicability was successfully validated by determining chloroacetamide herbicides in the corn sample with good recovery rates (85.4 to 109.3%) that correlate well with the regression model. The newly developed dual-mode LFIA with reduced detection time (12 min) holds great potential for pesticide monitoring in equipment-limited environments using a portable test strip reader and laboratory conditions using ML algorithms.

Ultrasensitive and Wash-Free Detection of Tumor Extracellular Vesicles by Aptamer-Proximity-Ligation-Activated Rolling Circle Amplification Coupled to Single Particle ICP-MS.

Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate measurement of TEVs presents challenges due to their low abundance and potential interference from a high number of EVs derived from normal cells. Herein, an aptamer-proximity-ligation-activated rolling circle amplification (RCA) method for EV membrane recognition, coupled with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the quantification of TEVs, is developed. When DNA-labeled ultrasmall gold nanoparticle (AuNP) probes bind to the long chains formed by RCA, they aggregate to form large particles. Notably, small AuNPs scarcely produce pulse signals in sp-ICP-MS, thereby detecting TEVs in a wash-free manner. By leveraging the strong binding affinity of aptamers, dual aptamers for EpCAM and PD-L1 recognition, and the sp-ICP-MS technique, this method offers remarkable sensitivity and selectivity in tracing TEVs. Under optimized conditions, the present method shows a favorable linear relationship between the pulse signal frequency of sp-ICP-MS and TEV concentration within the range of 105-107 particles/mL, along with a detection limit of 1.1 × 104 particles/mL. The pulse signals from sp-ICP-MS combined with machine learning algorithms are used to discriminate cancer patients from healthy donors with 100% accuracy. Due to its simple and fast operation and excellent sensitivity and accuracy, this approach holds significant potential for diverse applications in life sciences and personalized medicine.

3D-Printed SERS Chips for Highly Specific Detection of Denatured Type I and IV Collagens in Blood for Early Hepatic Fibrosis Diagnosis.

Hepatic fibrosis, the insidious progression of chronic liver scarring leading to life-threatening cirrhosis and hepatocellular carcinoma, necessitates the urgent development of noninvasive and precise diagnostic methodologies. Denatured collagen emerges as a critical biomarker in the pathogenesis of hepatic fibrosis. Herein, we have for the first time developed 3D-printed collagen capture chips for highly specific surface-enhanced Raman scattering (SERS) detection of denatured type I and type IV collagen in blood, facilitating the early diagnosis of hepatic fibrosis. Employing a novel blend of denatured collagen-targeting peptide-modified silver nanoparticle probes (Ag@DCTP) and polyethylene glycol diacrylate (PEGDA), we engineered a robust ink for the 3D fabrication of these collagen capture chips. The chips are further equipped with specialized SERS peptide probes, Ag@ICTP@R1 (S-I) and Ag@IVCTP@R2 (S-IV), tailored for the targeted detection of type I and IV collagen, respectively. The SERS chip platform demonstrated exceptional specificity and sensitivity in capturing and detecting denatured type I and IV collagen, achieving detection limits of 3.5 ng/mL for type I and 3.2 ng/mL for type IV collagen within a 10-400 ng/mL range. When tested on serum samples from hepatic fibrosis mouse models across a spectrum of fibrosis stages (S0-S4), the chips consistently measured denatured type I collagen and detected a progressive increase in type IV collagen concentration, which correlated with the severity of fibrosis. This novel strategy establishes a benchmark for the multiplexed detection of collagen biomarkers, enhancing our capacity to assess the stages of hepatic fibrosis.

Label-free Colorimetric Detection of Viral RNA Based on Clustered Regularly Interspaced Short Palindromic Repeats and Gold Nanoparticles with a Portable Device.

Viral infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are some of the most dangerous threats to humans. SARS-CoV-2 has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. To meet these requirements, we designed a label-free colorimetric platform that combines the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) 12a system for naked-eye detection (named LFP). This method utilizes reverse transcription loop-mediated isothermal amplification (RT-LAMP) and the trans-cleavage activity of the CRISPR/Cas12a system to increase the sensitivity and specificity of the reaction. This platform can detect as few as 4 copies/μL of RNA and produces no false positive results when tested against the influenza virus. To better meet the requirements of point-of-care (POC) detection, we developed a portable device that can be applied in resource-poor and densely populated regions. The LFP assay holds great potential for application in resource-limited settings, and the label-free gold nanoparticle (AuNPs) probe can reduce costs, making it suitable for large-scale screening. We expect that the LFP assay will be promising for the POC screening of COVID-19.

Two-Photon Nanoparticle Probe for Formaldehyde Detection via the AILE Luminescence Mechanism.

A two-photon nanoparticle probe was designed and developed based on the principle of intermolecular interaction of the aggregation-induced locally excited emission luminescence mechanism. The probe has the advantages of simple synthesis, convenient use, strong atomic economy, good biocompatibility, and photobleaching resistance. It can produce a specific and sensitive response to formaldehyde, help detect FA in normal cells and cancer cells, and is expected to become a specific detection probe for FA in vitro and in vivo.

Recent advances in molecular and nanoparticle probes for fluorescent bioanalysis

Recent advances in molecular and nanoparticle probes for fluorescent bioanalysis

Smartphone-Integrated resorcinarene macrocycle capped silver nanoparticles (RMF-AgNPs) probe for enhanced La(III) detection in diverse environments

The demand for precise and responsive detection of Lanthanum (La(III)) is critical in key areas such as environmental monitoring, biomedical research, and industrial processes, underscoring the need for advanced sensor technologies. This research reports an efficient and cost-effective preparation of resorcinarene macrocycle framework (RMF) capped silver nanoparticles (RMF-AgNPs). These nanoparticles were then applied as a novel sensor for La(III), demonstrating dual functionality: they act as a colorimetric sensor and a paper-based probe. Unique in its class, the sensor excels in detecting La(III) at ultra-low concentrations, unaffected by other ions, and undergoes a significant color transition from yellow to grey in response to La(III) presence. This change is linked to the localized surface plasmon resonance (LSPR) absorbance alteration. Extensive analytical methods like FTIR, XPS, AFM, FESEM, DLS, Zeta potential, and UV–Vis spectroscopy confirmed that the sensor operates by aggregating nanoparticles induced by La(III). Adding to its practicality, we incorporated a smartphone interface with the paper-based sensor, enhancing its utility for on-the-spot, real-time detection. The sensor's efficacy was validated across various samples, including soil, rock, urine, drinking water, and industrial effluent, showing its versatility. With detection limits of 15 nM (colorimetric) and 280 nM (paper-based), and linear dynamic ranges from 0.05 to 100 µM and 0.5–100 µM respectively, this sensor marks a significant advancement, effectively bridging lab-based detection with field application needs.

Gold nanoparticles-based colorimetric method for the rapid and sensitive detection of sour rot in onions

Onion (Allium cepa L.) is a major vegetable used in food preparation. It can also act as a medicinal compound for many diseases. However, various plant diseases, including sour rot, caused by the bacterium Burkholderia cepacia, is a post-harvest disease that can cause significant economic losses in onions and are considered a major threat to the onion industry. This study was performed to compare two methods for detecting sour rot: multiplex PCR and colorimetry with gold nanoparticles probe. Multiplex PCR used, primers designed for RecA, APZ15-03995 and APZ15-27735 genes of B. cepacia, and the results showed 100% specificity and sensitivity of 74.4 × 10−4 ng/μL of genomic DNA. Colorimetry with gold nanoparticles probe was performed using individual gene probes or a combination of three gene probes as a multi-colorimetric assay. Results showed 100% specificity and sensitivity of 74.4 × 10−8 ng/μL of genomic DNA in presence of the three probes. The results showed that both methods are specific and sensitive enough to detect B. cepacia. However, the use of gold nanoparticles is a more suitable option due to its high specificity and sensitivity and shorter time compared to multiplex PCR. Consequently, accurate and timely molecular identification of onion rot helps reduction of post-harvest losses and prevent the spread of secondary bacteria in storage.

Pullulan-Based Spray-Dried Mucoadhesive Microparticles for Sustained Oromucosal Drug Delivery.

Mucoadhesive microparticles for oromucosal drug delivery offer several advantages, including intimate contact with the mucosa, delivery to less accessible regions, extended residence time, sustained drug release, reduced irritation, and improved patient compliance. In this study, pullulan was used to prepare mucoadhesive spray-dried microparticles for delivering benzydamine hydrochloride (BZH) to oral mucosa. The BZH-pullulan spray-dried microparticles had a mean size of <25 μm with an angle of repose values between 25.8-36.6°. Pullulan markedly extended drug-release time to >180 min, ~9 times greater than the duration (i.e., 20 min) reportedly achieved by chitosan. Kinetic analysis showed the drug-release rate was concentration dependent and jointly controlled by drug diffusion and polymer chain relaxation. Further, pullulan was mucoadhesive and was able to retain up to 78.8% w/w of microencapsulated gold nanoparticle probes at the mucosal membrane. These data strongly suggest that BZH-pullulan microparticles have great potential for oromucosal drug delivery, by providing elongated residence time in situ and sustained drug release for the treatment of local diseases.

Magnetic nanoparticles coated with a molecularly imprinted polymer via manganese-doped ZnS:Mn quantum dots for the determination of Triflumuron

ABSTRACT A novel multifunctional silica magnetic molecularly imprinted polymer (MQDs-MIPs) nanoparticle probe was synthesized for the rapid detection of triflumuron (TFM). The fluorescence probe consists of magnetite (Fe3O4) as a core and is wrapped with a molecularly imprinted polymer doped with Mn-doped ZnS quantum dots (QDs). Under optimal detection condition, the fluorescence quenching efficiency of MIP@Fe3O4@ZnS:Mn QDs displayed a linear relationship over the concentration range of 0–27.88 μmol/L with a correlation coefficient of 0.9947. Four structurally similar compounds were selected for selectivity testing and the nanocomposite showed good selectivity with a higher imprint factor of 2.52. This developed method has the advantages of simple preparation, fast response and low toxicity for trace TFM detection.

Digital Surface-Enhanced Raman Spectroscopy-Lateral Flow Test Dipstick: Ultrasensitive, Rapid Virus Quantification in Environmental Dust.

This study introduces a novel surface-enhanced Raman spectroscopy (SERS)-based lateral flow test (LFT) dipstick that integrates digital analysis for highly sensitive and rapid viral quantification. The SERS-LFT dipsticks, incorporating gold-silver core-shell nanoparticle probes, enable pixel-based digital analysis of large-area SERS scans. Such an approach enables ultralow-level detection of viruses that readily distinguishes positive signals from background noise at the pixel level. The developed digital SERS-LFTs demonstrate limits of detection (LODs) of 180 fg for SARS-CoV-2 spike protein, 120 fg for nucleocapsid protein, and 7 plaque forming units for intact virus, all within <30 min. Importantly, digital SERS-LFT methods maintain their robustness and their LODs in the presence of indoor dust, thus underscoring their potential for accurate and reliable virus diagnosis and quantification in real-world environmental settings.