Biomimetic affinity sensor for the ultrasensitive detection of neonicotinoids.

Multiple pesticides are often used in combination to protect crops from pests. This makes rapid on-site detection of pesticide contamination challenging. Herein, we describe a method for simultaneous detection of diverse neonicotinoid pesticides using a sensor that combines neonicotinoid-specific odorant-binding protein 2 (OBP2), which was cloned from an insect chemical sensing protein and modified gold nanoparticles with local surface plasmon resonance (LSPR)-based digital nanoplasmonometry (DiNM). When neonicotinoid pesticides bind to OBP2 on gold nanoparticles, the induced LSPR shift peak wavelength is too small to be measured using conventional LSPR immunoassays. DiNM records and compares the scattered image intensity in two adjacent wavelength bands, A and B, centered on the LSPR peak. It considers both the peak shift and the relative intensity change in these two bands, resulting in a significant LSPR signal enhancement. Then the spectral-image contrast was computed as the signal response. Using this approach, we obtained excellent limits of detection (LODs) of 1.4, 1.5, and 4.5 ppb for the neonicotinoids imidacloprid, acetamiprid, and dinotefuran, respectively. Blind tests demonstrated high positive and negative rates for teas, approximately 85 and 100%, respectively. Recombinant OBP2 produced in E. coli offers several advantages over antibodies, including high yield, time savings, and cost effectiveness. Moreover, this method is highly selective and sensitive to neonicotinoids, making it practical for field use.

Dual Gold-Nanoslit Electrodes for Ultrasensitive Detection of Antigen-Antibody Reactions in Electrochemical Surface Plasmon Resonance.

We present the use of surface charges in dual gold-nanoslit electrodes to improve the surface plasmon resonance (SPR) detection limit by several orders of magnitude. The SPR is directly generated by gold-nanoslit arrays in the two electrodes. The SPR shifts for both nanoslit arrays are measured simultaneously with a simple hyperspectral setup. When biomolecules are captured by specific antibodies on the dual electrodes, the surface charge is changed during the electrochemical process due to the increase in surface impedance. The push-pull-type electrodes generate opposite surface charges. Using the differences in both spectral shifts, the change in surface charge is detected sensitively. We demonstrate that using a [Fe(CN)6]3-/4- redox process after antigen-antibody interactions, the dual nanoslit electrodes show an enhancement of the detection limit from 1 µg/mL to 10 pg/mL.

Sensitive Oligonucleotide Detection Using Resonant Coupling between Fano Resonance and Image Dipoles of Gold Nanoparticles.

The surface plasmon resonance (SPR)-based sensor has been widely used for biodetection. One of the attractive roles is the gold nanostructure with Fano resonance. Its sharp resonant profile takes advantage of the high figure of merit (FoM) in high-sensitivity detection. However, it is still difficult to detect small molecules at low concentrations due to the extremely low refractive index changes on the metallic surface. We propose using the coupling of image dipoles of gold nanoparticles (AuNPs) and Fano resonance of periodic capped gold nanoslits (CGNs) for sensitive small-molecule detections. The coupling mechanism was verified by three-dimensional finite-difference time-domain calculations and experiments. AuNPs on CGN form image dimer assemblies and induce image dipole with resonance wavelengths ranging from 730 to 550 nm. The surface plasmon polaritons (SPPs) interact with the image dipole of the AuNP on the CGNs and then scatter out through the periodic gold caps. The experimental results show that the peak intensity of grating resonance is decreased by the effect of image dipole and exhibits the maximum intensity change when the Fano resonance matches the resonance of image dipole. The 50 nm AuNPs can be detected with a surface density of less than one particle/µm2 by using the intensity change as the signal. With the resonant coupling between Fano resonance and image dipole extinction, the oligonucleotide with a molecular weight of 5.5 kDa can be detected at a concentration of 100 fM. The resonant coupling dramatically pushes the sensitivity boundary, and we report the limit of detection (LOD) to be 3 orders of magnitude lower than that of the prism-based SPR. This study provides a promising and efficient method for detecting low concentrations of small molecules such as aptamers, miRNA, mRNA, and peptides.

Spectral image contrast-based flow digital nanoplasmon-metry for ultrasensitive antibody detection.

BACKGROUND: Gold nanoparticles (AuNPs) have been widely used in local surface plasmon resonance (LSPR) immunoassays for biomolecule sensing, which is primarily based on two conventional methods: absorption spectra analysis and colorimetry. The low figure of merit (FoM) of the LSPR and high-concentration AuNP requirement restrict their limit of detection (LOD), which is approximately ng to µg mL-1 in antibody detection if there is no other signal or analyte amplification. Improvements in sensitivity have been slow in recent for a long time, and pushing the boundary of the current LOD is a great challenge of current LSPR immunoassays in biosensing. RESULTS: In this work, we developed spectral image contrast-based flow digital nanoplasmon-metry (Flow DiNM) to push the LOD boundary. Comparing the scattering image brightness of AuNPs in two neighboring wavelength bands near the LSPR peak, the peak shift signal is strongly amplified and quickly detected. Introducing digital analysis, the Flow DiNM provides an ultrahigh signal-to-noise ratio and has a lower sample volume requirement. Compared to the conventional analog LSPR immunoassay, Flow DiNM for anti-BSA detection in pure samples has an LOD as low as 1 pg mL-1 within only a 15-min detection time and 500 µL sample volume. Antibody assays against spike proteins of SARS-CoV-2 in artificial saliva that contained various proteins were also conducted to validate the detection of Flow DiNM in complicated samples. Flow DiNM shows significant discrimination in detection with an LOD of 10 pg mL-1 and a broad dynamic detection range of five orders of magnitude. CONCLUSION: Together with the quick readout time and simple operation, this work clearly demonstrated the high sensitivity and selectivity of the developed Flow DiNM in rapid antibody detection. Spectral image contrast and digital analysis further provide a new generation of LSPR immunoassay with AuNPs.

Polyglutamine-Specific Gold Nanoparticle Complex Alleviates Mutant Huntingtin-Induced Toxicity.

Huntington's disease (HD) belongs to protein misfolding disorders associated with polyglutamine (polyQ)-rich mutant huntingtin (mHtt) protein inclusions. Currently, it is indicated that the aggregation of polyQ-rich mHtt participates in neuronal toxicity and dysfunction. Here, we designed and synthesized a polyglutamine-specific gold nanoparticle (AuNP) complex, which specifically targeted mHtt and alleviated its toxicity. The polyglutamine-specific AuNPs were prepared by decorating the surface of AuNPs with an amphiphilic peptide (JLD1) consisting of both polyglutamine-binding sequences and negatively charged sequences. By applying the polyQ aggregation model system, we demonstrated that AuNPs-JLD1 dissociated the fibrillary aggregates from the polyQ peptide and reduced its ß-sheet content in a concentration-dependent manner. By further integrating polyethyleneimine (PEI) onto AuNPs-JLD1, we generated a complex (AuNPs-JLD1-PEI). We showed that this complex could penetrate cells, bind to cytosolic mHtt proteins, dissociate mHtt inclusions, reduce mHtt oligomers, and ameliorate mHtt-induced toxicity. AuNPs-JLD1-PEI was also able to be transported to the brain and improved the functional deterioration in the HD Drosophila larva model. Our results revealed the feasibility of combining AuNPs, JLD1s, and cell-penetrating polymers against mHtt protein aggregation and oligomerization, which hinted on the early therapeutic strategies against HD.

Self-referencing biosensors using Fano resonance in periodic aluminium nanostructures.

Surface plasmon resonance (SPR) is an important technique for real-time and label-free detection of specific binding biomolecules. However, conventional SPR signals come from both the surface binding biomolecules and the variation in the bulk refractive index. This work demonstrates that Fano resonance in an aluminum capped nanoslit array has the ability to remove the signal of bulk refractive index changes from the SPR signal. As compared to gold nanostructures, the aluminum nanostructure provides an asymmetrical Fano resonance with clear peak and dip wavelengths. The peak wavelength is close to the grating resonance condition. The evanescent depth at the peak wavelength is up to several microns. The dip wavelength comes from the SPR effect. The evanescent depth at the dip wavelength is about 300 nm. By simultaneously measuring the shifts of peaks and the dip wavelengths, the variation in the bulk refractive index can be removed and only the biolayer thickness is measured. The finite-difference time-domain calculation shows that the 470 nm-period nanoslit array with 90 and 70 nm slit depths has the optimal thickness sensitivity. In this experiment, a simple multispectral imaging system is developed for multiple bio-interaction measurements. The measured results verify that the bulk refractive index changes can be removed and the surface biomolecular interactions can be directly obtained without the need of a reference channel.

Dispersion-enhancing surface treatment of AuNPs for a reduced probe loading and detection limit using t-SPR detection.

COVID-19 has shown that a highly specific and rapid diagnostic system is a necessity. A spectral imaging-based surface plasmon resonance (SPRi) platform with an integrated microfluidic biosensor to detect oligonucleotide sequences has been proposed to be a promising alternative for infectious diseases due to its safe and straightforward use. Approaches to reduce the DNA probe loading onto gold nanoparticles with various types of polyethylene glycol (PEG) were explored. Here, we demonstrated the stability of functionalised gold nanoparticles with unmodified PEG whilst lowering the probe loading density. The system was evaluated by performing the detection of a mimicking COVID-19 target sequence, single point-mutation sequence and fully mismatch sequence. Highly specific binding of the mimicking COVID-19 target sequence was observed and analysed by the spectral imaging SPR approach. Our work has demonstrated the potential of a controlled probe density using unmodified PEG as an especially promising functionalisation strategy in SPR spectral imaging assays.

Enhancing Raman signals from bacteria using dielectrophoretic force between conductive lensed fiber and black silicon.

An osmium-coated lensed fiber (OLF) probe combined with a silver-coated black silicon (SBS) substrate was used to generate a dielectrophoretic (DEP) force that traps bacteria and enables Raman signal detection from bacteria. The lensed fiber coated with a 2-nm osmium layer was used as an electrode for the DEP force and also as a lens to excite Raman signals. The black silicon coated with a 150-nm silver layer was used both as the surface-enhanced Raman scattering (SERS) substrate and the counter electrode. The enhanced Raman signal was collected by the same OLF probe and further analyzed with a spectrometer. For Raman measurements, a drop of bacterial suspension was placed between the OLF probe and the SBS substrate. By controlling the frequency of an AC voltage on the OLF probe and SBS substrate, a DEP force at 1 MHz concentrated bacteria on the SBS surface and removed the unbound micro-objects in the solution at 1 kHz. A bacteria concentration of 6 × 104 CFU/mL (colony forming units per mL) could be identified in less than 15 min, using a volume of only 1 µL, by recording the variation of the Raman peak at 740 cm-1.

Multichannel nanoplasmonic platform for imidacloprid and fipronil residues rapid screen detection.

In recent years, imidacloprid and fipronil have been reported to harm beneficial insects, such as honey bees, and to potentially pose risks to mammals, including humans. Considering their widespread use and potential minimum toxic range from 10 ppb to 1 ppm (species dependent), a simple, rapid, sensitive, and reliable method for screening and detection is urgently needed. Here, we present a surface plasmon resonance (SPR)-based nanoplasmonic chip integrated with a multichannel spectral imaging system to detect ecosystem-harming pesticides. The pre-modification of the designed mercapto-haptens reduced detection time to 2.5 h. Moreover, owing to the multichannel configuration, it was possible to introduce an internal standard analytical method to effectively reduce matrix interference in real samples; thus, the concentration of the target pesticide could be determined more precisely. The strong linearity of the spiked sample test results indicated high accuracy in quantifying target pesticides. Considering the limit of detection (~10 ppb), the cutoffs for detection and quantification were set at 15 and 45 ppb, respectively, and were used as the detection criteria. The detection results of the blind tests of real samples were also compared with those of liquid chromatography electrospray ionization tandem mass spectrometry (standard method) and were highly consistent. The custom-made integrated SPR system allows much simpler, label-free, high-throughput, and reliable on-site identification and quantification of imidacloprid and fipronil. All test results validated the platform's capability in the on-site rapid screening and detection of pesticide residues at the parts per billion and parts per million levels.

Evolution of gold nanoparticle clusters in living cells studied by sectional dark-field optical microscopy and chromatic analysis.

The evolution of gold nanoparticle (Au NP) clusters in living cells are studied by using sectional dark-field optical microscopy and chromatic analysis approach. During endocytosis, Au NP clusters undergo fantastic color changes, from green to yellow-orange due to the plasmonic coupling effect. Analysis of brightness/hue values of the dark-field images helps estimate the numbers of Au NPs in the clusters. The Au NP clusters were further categorized into four groups within the endocytosis. As the results, the late endosomes had increased number of large Au NP clusters with time, while clustered numbers in secondary and tertiary groups were first increased and then decreased due to the fusion and fission of the endocytic vesicles. The time constants and cluster numbers for different groups are fitted by using an integrated rate equation, and show a positive correlation with the size of the Au NP cluster. The efficiency of Au NP uptake is only about 50% for normal cells, while 75% for cancer cells. Compared to normal cells, cancer cells show a larger number in uptake, while faster rate in removal. The propose method helps the kinetic study of endocytosed nanoparticles in physiological conditions.

Size-dependent endocytosis of gold nanoparticles studied by three-dimensional mapping of plasmonic scattering images.

BACKGROUND: Understanding the endocytosis process of gold nanoparticles (AuNPs) is important for the drug delivery and photodynamic therapy applications. The endocytosis in living cells is usually studied by fluorescent microscopy. The fluorescent labeling suffers from photobleaching. Besides, quantitative estimation of the cellular uptake is not easy. In this paper, the size-dependent endocytosis of AuNPs was investigated by using plasmonic scattering images without any labeling. RESULTS: The scattering images of AuNPs and the vesicles were mapped by using an optical sectioning microscopy with dark-field illumination. AuNPs have large optical scatterings at 550-600 nm wavelengths due to localized surface plasmon resonances. Using an enhanced contrast between yellow and blue CCD images, AuNPs can be well distinguished from cellular organelles. The tracking of AuNPs coated with aptamers for surface mucin glycoprotein shows that AuNPs attached to extracellular matrix and moved towards center of the cell. Most 75-nm-AuNPs moved to the top of cells, while many 45-nm-AuNPs entered cells through endocytosis and accumulated in endocytic vesicles. The amounts of cellular uptake decreased with the increase of particle size. CONCLUSIONS: We quantitatively studied the endocytosis of AuNPs with different sizes in various cancer cells. The plasmonic scattering images confirm the size-dependent endocytosis of AuNPs. The 45-nm-AuNP is better for drug delivery due to its higher uptake rate. On the other hand, large AuNPs are immobilized on the cell membrane. They can be used to reconstruct the cell morphology.