Direct detection of virus-like particles using color images of plasmonic nanostructures.

We propose a measurement method for sensitive and label-free detections of virus-like particles (VLPs) using color images of nanoplasmonic sensing chips. The nanoplasmonic chip consists of 5×5 gold nanoslit arrays and the gold surface is modified with specific antibodies for spike protein. The resonant wavelength of the 430-nm-period gold nanoslit arrays underwater environment is about 570 nm which falls between the green and red bands of the color CCD. The captured VLPs by the specific antibodies shift the plasmonic resonance of the gold nanoslits. It results in an increased brightness of green pixels and decreased brightness of red pixels. The image contrast signals of (green - red) / (red + green) show good linearity with the surface particle density. The experimental tests show the image contrast method can detect 100-nm polystyrene particles with a surface density smaller than 2 particles/µm2. We demonstrate the application for direct detection of SARS-CoV-2 VLPs using a simple scanner platform. A detection limit smaller than 1 pg/mL with a detection time less than 30 minutes can be achieved.

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.

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.

Screening anti-metastasis drugs by cell adhesion-induced color change in a biochip.

Metastasis is a frequent complication of cancer and accounts for more than 60% of patients' mortality. Despite technological advancements, treatment options are still limited. Ion channels participate in the regulation of cell adhesion, whilst the regulation of cell adhesion further controls metastasis formation. However, to develop a new ion channel inhibitor targeting metastasis takes tremendous effort and resources; therefore, drug repurposing is an emerging strategy in oncology. In previous studies, we have developed a metal-based nanoslit surface plasmon resonance (SPR) platform to examine the influence of drugs on the cell adhesion process. In this work, we developed a scanner-based cell adhesion kinetic examination (CAKE) system that is capable of monitoring the cell adhesion process by measuring color changes of SPR biosensors. The system's performance was demonstrated by screening the anti-metastasis ability of compounds from a commercial ion-channel inhibitor library. Out of the 274 compounds from the inhibitor library, zinc pyrithione (ZPT) and terfenadine were demonstrated to influence CL1-5 cell adhesion. The cell responses to the two compounds were then compared with those by traditional cell adhesion assays where similar behavior was observed. Further investigation of the two compounds using wound healing and transwell assays was performed and inhibitions of both cell migration and invasion by the two compounds were also observed. The results indicate that ZPT and terfenadine are potential candidates for anti-metastasis drugs. Our work has demonstrated the label-free drug screening ability of our CAKE system for finding potential drugs for cancer treatment.

Large shift of resonance wavelengths of silver nanoslit arrays using electrowetting-on-dielectric cells.

A simple design for shifting the resonance wavelength of silver nanoslits using an electrowetting-on-dielectric (EWOD) cell is proposed. The EWOD cell comprises a polycarbonate (PC) substrate with Teflon-coated silver nanoslits and a glass substrate with Teflon-coated electrodes. A glycerol droplet is placed between the two substrates, and out of the path of a probe beam at zero electric field. Application of an electric field smaller than 0.3 V/µm on the electrodes moves the glycerol droplet into the path of the probe beam, shifting the resonance wavelength of the silver nanoslits by 135 nm. A change (0.33) in the refractive index of the effective medium that is adjacent to the silver nanoslits causes a large shift in the resonance wavelength. The spectral shift of the silver nanoslits is repeatable by the electric field. This simple design is a great achievement for high-performance electro-optical devices with large wavelength shift ranges such as optical switches, variable optical attenuators, and sensor applications.

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.

High-Throughput and Dynamic Study of Drug and Cell Interactions Using Contrast Images in Aluminum-Based Nanoslit Arrays.

High-throughput and dynamic measurement for living cell activities can benefit biological research and drug development. A low-cost metallic nanostructure-based surface plasmon resonance (SPR) imaging platform, comprising multiple aluminum nanoslit arrays and a color image device, is proposed for label-free study of cell and drug interactions. The multiple nanoslit sensing arrays were fabricated using the compression-injection molding process. These sensing chips showed a detectable depth of 600 nm and refractive index resolution of ∼5 × 10-5 refractive index unit (RIU) by using a self-referenced two-color analysis. Two examples of kinetic studies of living cells under various doses of drugs are presented. The focal adhesion kinases inhibitor (FAKi 14) and cell interactions show exponential changes of cellular adhesion and time constants for different concentrations of antiadhesion drugs. The anticancer drug (doxorubicin (DOX))-treated cells show slow increases of SPR signals in the first 2 h due to the nucleus swelling. The DOX-treated cells further process plasma membrane disruption and become floating cells and debris in the medium, resulting in rapid drops of the SPR signals.

Spectral contrast imaging method for mapping transmission surface plasmon images in metallic nanostructures.

We propose a spectral contrast method to map the transmission images of surface plasmon resonance (SPR) in metallic nanostructures. Comparing the intensities between two neighboring wavelength bands near the SPR wavelength, the signal-to-noise ratio for biosensing applications obtained using the proposed method is found to be ten times higher than that obtained by conventional intensity analysis and 1.6 times better than that obtained by peak-wavelength fitting. The dynamic range and linearity of the refractive index are comparable to the peak-wavelength shift measurement. Based on the detection method, a spectral modulation system for the optical microscope is developed, combined with a gold-capped nanowire array, to measure the biointeractions in microfluidic devices. The experimental results show that the proposed method obtained multiple detections with a detection limit of 1.04 × 10-5 refractive index units. Two types of analysis methods for SPR images are used to study the protein-antibody interactions. The region-of-interest analysis supports multiplexing detections in a compact microfluidic sensor. The effective pixel analysis eliminates low-response pixels and enhances the signal-to-noise ratios for sensitive label-free detection.

Simultaneous assessment of cell morphology and adhesion using aluminum nanoslit-based plasmonic biosensing chips.

A variety of physiological and pathological processes rely on cell adhesion, which is most often tracked by changes in cellular morphology. We previously reported a novel gold nanoslit-based biosensor that is capable of real-time and label-free monitoring of cell morphological changes and cell viability. However, the preparation of gold biosensors is inefficient, complicated and costly. Recently, nanostructure-based aluminum (Al) sensors have been introduced for biosensing applications. The Al-based sensor has a longer decay length and is capable of analyzing large-sized mass such as cells. Here, we developed two types of double-layer Al nanoslit-based plasmonic biosensors, which were nanofabricated and used to evaluate the correlation between metastatic potency and adhesion of lung cancer and melanoma cell lines. Cell adhesion was determined by Fano resonance signals that were induced by binding of the cells to the nanoslit. The peak and dip of the Fano resonance spectrum respectively reflected long- and short-range cellular changes, allowing us to simultaneously detect and distinguish between focal adhesion and cell spreading. Also, the Al nanoslit-based biosensor chips were used to evaluate the inhibitory effects of drugs on cancer cell spreading. We are the first to report the use of double layer Al nanoslit-based biosensors for detection of cell behavior, and such devices may become powerful tools for anti-metastasis drug screening in the future.

Multiplex detection of urinary miRNA biomarkers by transmission surface plasmon resonance.

The clinical assessment of short-stranded nucleic acid biomarkers such as miRNAs could potentially provide useful information for monitoring disease progression, prompting definitive treatment decisions. In the past decade, advancements in biosensing technology have led to a shift towards rapid, real-time and label-free detection systems; as such, surface plasmon resonance (SPR) biosensor-based technology has become of high interest. Here, we developed an automated multiplex transmissive surface plasmon resonance (t-SPR) platform with the use of a capped gold nanoslit integrated microfluidic surface plasmon resonance (SPR) biosensor. The automated platform was custom designed to allow the analysis of spectral measurements using wavelength shift (dλ), intensity (dI) and novel area change (dA) for surface binding reactions. A simple and compact nanostructure based biosensor was fabricated with multiplex real-time detection capabilities. The sensitivity and specificity of the microfluidic device was demonstrated through the use of functionalised AuNPs for target molecule isolation and signal enhancement in combination with probes on the CG nanoslit surface. Our work allows for the multiplex detection of miRNA at femtomolar concentrations in complex media such as urine.

Resonant position tracking method for smartphone-based surface plasmon sensor.

We propose a position-sensitive measurement method for tracking resonant signals of surface plasmon resonance (SPR) in periodic metallic nanostructures. Compared with conventional measurement methods, such as wavelength interrogation, intensity interrogation and spectral integration, this approach provides superior noise reduction and simple calculation process. Experimental results show that the limit of detection reaches to 5.88 × 10-6 RIU by using a portable spectrometer with a spectral resolution of 0.4 nm. The relationship between shot noise and signal noise was theoretically compared. The superior noise reduction of the resonant position tracking method is useful for the smartphone-based SPR measurement. The protein-antibody interactions using the smartphone and gold nanoslit arrays as the SPR sensor are demonstrated. It verifies that a smartphone can be used for sensitive measurement of binding-kinetics of the proteins.

Spectral Imaging Analysis for Ultrasensitive Biomolecular Detection Using Gold-Capped Nanowire Arrays.

A spectral integration combined with a threshold method for the analysis of spectral scanning surface plasmon resonance (SPR) images can significantly increase signal recognition at low concentration of antibody solution. The 12-well SPR sensing plates consisted of gold-capped nanowire arrays with 500-nm period, 80-nm linewidth and 50-nm gold thickness which were used for generating multiple SPR images. A threshold method is introduced to eliminate background noises in spectral scanning images. Combining spectral integration and the threshold method, the detection limit of antibody concentration was 1.23 ng/mL. Using multiple-well SPR sensing plates and the proposed analytical method, multiple kinetic responses with spectral and spatial information on different sensing areas can be sensitively measured.

Enhancing Surface Sensing Sensitivity of Metallic Nanostructures using Blue-Shifted Surface Plasmon Mode and Fano Resonance.

Improving surface sensitivities of nanostructure-based plasmonic sensors is an important issue to be addressed. Among the SPR measurements, the wavelength interrogation is commonly utilized. We proposed using blue-shifted surface plasmon mode and Fano resonance, caused by the coupling of a cavity mode (angle-independent) and the surface plasmon mode (angle-dependent) in a long-periodicity silver nanoslit array, to increase surface (wavelength) sensitivities of metallic nanostructures. It results in an improvement by at least a factor of 4 in the spectral shift as compared to sensors operated under normal incidence. The improved surface sensitivity was attributed to a high refractive index sensitivity and the decrease of plasmonic evanescent field caused by two effects, the Fano coupling and the blue-shifted resonance. These concepts can enhance the sensing capability and be applicable to various metallic nanostructures with periodicities.

Low-Cost and Rapid Fabrication of Metallic Nanostructures for Sensitive Biosensors Using Hot-Embossing and Dielectric-Heating Nanoimprint Methods.

We propose two approaches-hot-embossing and dielectric-heating nanoimprinting methods-for low-cost and rapid fabrication of periodic nanostructures. Each nanofabrication process for the imprinted plastic nanostructures is completed within several seconds without the use of release agents and epoxy. Low-cost, large-area, and highly sensitive aluminum nanostructures on A4 size plastic films are fabricated by evaporating aluminum film on hot-embossing nanostructures. The narrowest bandwidth of the Fano resonance is only 2.7 nm in the visible light region. The periodic aluminum nanostructure achieves a figure of merit of 150, and an intensity sensitivity of 29,345%/RIU (refractive index unit). The rapid fabrication is also achieved by using radio-frequency (RF) sensitive plastic films and a commercial RF welding machine. The dielectric-heating, using RF power, takes advantage of the rapid heating/cooling process and lower electric power consumption. The fabricated capped aluminum nanoslit array has a 5 nm Fano linewidth and 490.46 nm/RIU wavelength sensitivity. The biosensing capabilities of the metallic nanostructures are further verified by measuring antigen-antibody interactions using bovine serum albumin (BSA) and anti-BSA. These rapid and high-throughput fabrication methods can benefit low-cost, highly sensitive biosensors and other sensing applications.

Highly Sensitive Aluminum-Based Biosensors using Tailorable Fano Resonances in Capped Nanostructures.

Metallic nanostructure-based surface plasmon sensors are capable of real-time, label-free, and multiplexed detections for chemical and biomedical applications. Recently, the studies of aluminum-based biosensors have attracted a large attention because aluminum is a more cost-effective metal and relatively stable. However, the intrinsic properties of aluminum, having a large imaginary part of the dielectric function and a longer evanescent length, limit its sensing capability. Here we show that capped aluminum nanoslits fabricated on plastic films using hot embossing lithography can provide tailorable Fano resonances. Changing height of nanostructures and deposited metal film thickness modulated the transmission spectrum, which varied from Wood's anomaly-dominant resonance, asymmetric Fano profile to surface plasmon-dominant resonance. For biolayer detections, the maximum surface sensitivity occurred at the dip of asymmetric Fano profile. The optimal Fano factor was close to -1.3. The wavelength and intensity sensitivities for surface thickness were up to 2.58 nm/nm and 90%/nm, respectively. The limit of detection (LOD) of thickness reached 0.018 nm. We attributed the enhanced surface sensitivity for capped aluminum nanoslits to a reduced evanescent length and sharp slope of the asymmetric Fano profile. The protein-protein interaction experiments verified the high sensitivity of capped nanostructures. The LOD was down to 236 fg/mL.

Chip-based digital surface plasmon resonance sensing platform for ultrasensitive biomolecular detection.

A chip-based ultrasensitive surface plasmon resonance (SPR) sensor in a checkerboard nanostructure on plastic substrates is presented for digital detection. The sensing elements on the checkerboard are composed of silver-capped nanoslit arrays, which were fabricated using the thermal-embossing nanoimprint method, to meet the demand for low-cost and rapid fabrication. Sharp Fano resonances in the optimized nanoslit arrays provide high-intensity sensitivities (20,000% per refractive index unit), with an element size of 12.5µm. The polarization-dependent transmission in the checkerboard pattern produces optical isolation between sensing elements and results in a crosstalk lower than 1%. Protein-antibody experiments demonstrated that the digital detection limit was up to 1pg/mL, which is approximately 1000 times lower than that of conventional analog detection. For a 140µm×140µm checkerboard pattern, the dynamic range was approximately 100 times higher than that of conventional surface plasmon resonance measurements. This new digital detection method is very useful for detecting ultralow concentrations of analytes with a nonuniform distribution on the sensor surface.

Enhancing Surface Sensitivity of Nanostructure-Based Aluminum Sensors Using Capped Dielectric Layers.

The studies of nanostructure-based aluminum sensors have attracted huge attention because aluminum is a more cost-effective plasmonic material. However, the intrinsic properties of the aluminum metal, having a large imaginary part of the dielectric function and a longer electromagnetic field decay length and problems of poor long-term chemical stability, limit the surface-sensing capability and applicability of nanostructures. We propose the combination of capped aluminum nanoslits and a thin-capped dielectric layer to overcome these limitations. We show that the dielectric layer can positively enhance the wavelength sensitivities of the Wood's anomaly-dominant resonance and asymmetric Fano resonance in capped aluminum nanoslits. The maximum improvement can be reached by a factor of 3.5. Besides, there is an optimal layer thickness for the surface sensitivity because of the trade-off relationship between the refractive index sensitivity and decay length. We attribute the enhanced surface sensitivity to a reduced evanescent length, which is confirmed by the finite difference time-domain calculations. The protein-protein interaction experiments verify the high-surface sensitivity of the structures, and a limit of quantification (LOQ) of 1 pg/mL anti-bovine serum albumin is achieved. Such low-cost, highly sensitive aluminum-based nanostructures can benefit various sensing applications.

Enhancing the Surface Sensitivity of Metallic Nanostructures Using Oblique-Angle-Induced Fano Resonances.

Surface sensitivity is an important factor that determines the minimum amount of biomolecules detected by surface plasmon resonance (SPR) sensors. We propose the use of oblique-angle-induced Fano resonances caused by two-mode coupling or three-mode coupling between the localized SPR mode and long-range surface plasmon polariton modes to increase the surface sensitivities of silver capped nanoslits. The results indicate that the coupled resonance between the split SPR (-kSPR) and cavity modes (two-mode coupling) has a high wavelength sensitivity for small-angle incidence (2°) due to its short decay length. Additionally, three-mode coupling between the split SPR (-kSPR), substrate (+kSub) and cavity modes has a high intensity sensitivity for large-angle incidence due to its short decay length, large resonance slope and enhanced transmission intensity. Compared to the wavelength measurement, the intensity measurement has a lower detectable (surface) concentration below 1 ng/ml (0.14 pg/mm(2)) and is reduced by at least 3 orders of magnitude. In addition, based on the calibration curve and current system noise, a theoretical detection limit of 2.73 pg/ml (0.38 fg/mm(2)) can be achieved. Such a surface concentration is close to that of prism-based SPR with phase measurement (0.1-0.2 fg/mm(2) under a phase shift of 5 mdeg).

Nanoplasmonic biochips for rapid label-free detection of imidacloprid pesticides with a smartphone.

The widespread and intensive use of neonicotinoid insecticides induces negative cascading effects on ecosystems. It is desirable to develop a portable sensitive sensing platform for on-site screening of high-risk pesticides. We combined an indirect competitive immunoassay, highly sensitive surface plasmon resonance (SPR) biochip and a simple portable imaging setup for label-free detection of imidacloprid pesticides. The SPR biochip consists of several capped nanoslit arrays with different periods which form a spectral image on the chip. The qualitative and semiquantitative analyses of pesticides can be directly observed from the spot shift on the chip. The precise semiquantitative analyses can be further completed by using image processing in a smartphone. We demonstrate simultaneous detection of four different concentrations of imidacloprid pesticides. The visual detection limit is about 1ppb, which is well below the maximum residue concentration permitted by law (20ppb). Compared to the one-step strip assay, the proposed chip is capable of performing semiquantitative analyses and multiple detection. Compared to the enzyme-linked immunosorbent assay, our method is label-free and requires simple washing steps and short reaction time. In addition, the label-free chip has a comparable sensitivity but wider working range than those labeling techniques.

Visualization of biosensors using enhanced surface plasmon resonances in capped silver nanostructures.

We propose a method and optical design for direct visualization of label-free detection. The system, similar to a tiny spectral analyzer, is composed of a nanostructure-based surface plasmon resonance chip, linear polarizer and 532 nm laser light source. The full-width-at-half-maximum bandwidths of the enhanced surface plasmon resonances are about 5 nm. The distribution of the transmitted light from these arrays comprises a spectral image on the chip. The qualitative and quantitative analyses of the analyte can be conducted by observing the spot shift on the chip. We tested the sensing capability of the chip. The detectable surface mass density with the naked eye is about 0.476 µg cm(-2). In addition, antigen-antibody interaction experiments are conducted to verify the surface binding measurements. A monolayer protein attached on the chip can be directly observed and the concentration levels of the analyte can be estimated with the naked eye. Such plasmonic biochips can benefit sensing applications in point-of-care diagnostics.