Optical Fingerprint of Flat Substrate Surface and Marker-Free Lateral Displacement Detection with Angstrom-Level Precision.

We report that flat substrates such as glass coverslips with surface roughness well below 0.5 nm feature notable speckle patterns when observed with high-sensitivity interference microscopy. We uncover that these speckle patterns unambiguously originate from the subnanometer surface undulations, and develop an intuitive model to illustrate how subnanometer nonresonant dielectric features could generate pronounced interference contrast in the far field. We introduce the concept of optical fingerprint for the deterministic speckle pattern associated with a particular substrate surface area and intentionally enhance the speckle amplitudes for potential applications. We demonstrate such optical fingerprints can be leveraged for reproducible position identification and marker-free lateral displacement detection with an experimental precision of 0.22 nm. The reproducible position identification allows us to detect new nanoscopic features developed during laborious processes performed outside of the microscope. The demonstrated capability for ultrasensitive displacement detection may find applications in the semiconductor industry and superresolution optical microscopy.

Nanoscopic Structural Fluctuations of Disassembling Microtubules Revealed by Label-Free Super-Resolution Microscopy.

Microtubules are cytoskeletal polymers of tubulin dimers assembled into protofilaments that constitute nanotubes undergoing periods of assembly and disassembly. Static electron micrographs suggest a structural transition of straight protofilaments into curved ones occurring at the tips of disassembling microtubules. However, these structural transitions have never been observed and the process of microtubule disassembly thus remains unclear. Here, label-free optical microscopy capable of selective imaging of the transient structural changes of protofilaments at the tip of a disassembling microtubule is introduced. Upon induced disassembly, the transition of ordered protofilaments into a disordered conformation is resolved at the tip of the microtubule. Imaging the unbinding of individual tubulin oligomers from the microtubule tip reveals transient pauses and relapses in the disassembly, concurrent with increased organization of protofilament segments at the microtubule tip. These findings show that microtubule disassembly is a discrete process and suggest a stochastic mechanism of switching from the disassembly to the assembly phase.

Fast Leaps between Millisecond Confinements Govern Ase1 Diffusion along Microtubules.

Diffusion is the most fundamental mode of protein translocation within cells. Confined diffusion of proteins along the electrostatic potential constituted by the surface of microtubules, although modeled meticulously in molecular dynamics simulations, has not been experimentally observed in real-time. Here, interferometric scattering microscopy is used to directly visualize the movement of the microtubule-associated protein Ase1 along the microtubule surface at nanometer and microsecond resolution. Millisecond confinements of Ase1 and fast leaps between these positions of dwelling preferentially occurring along the microtubule protofilaments are resolved, revealing Ase1's mode of diffusive translocation along the microtubule's periodic surface. The derived interaction potential closely matches the tubulin-dimer periodicity and the distribution of the electrostatic potential on the microtubule lattice. It is anticipated that mapping the interaction landscapes for different proteins on microtubules, finding plausible energetic barriers of different positioning and heights, can provide valuable insights into regulating the dynamics of essential cytoskeletal processes, such as intracellular cargo trafficking, cell division, and morphogenesis, all of which rely on diffusive translocation of proteins along microtubules.

Fast photothermal spatial light modulation for quantitative phase imaging at the nanoscale.

Spatial light modulators have become an essential tool for advanced microscopy, enabling breakthroughs in 3D, phase, and super-resolution imaging. However, continuous spatial-light modulation that is capable of capturing sub-millisecond microscopic motion without diffraction artifacts and polarization dependence is challenging. Here we present a photothermal spatial light modulator (PT-SLM) enabling fast phase imaging for nanoscopic 3D reconstruction. The PT-SLM can generate a step-like wavefront change, free of diffraction artifacts, with a high transmittance and a modulation efficiency independent of light polarization. We achieve a phase-shift > π and a response time as short as 70 µs with a theoretical limit in the sub microsecond range. We used the PT-SLM to perform quantitative phase imaging of sub-diffractional species to decipher the 3D nanoscopic displacement of microtubules and study the trajectory of a diffusive microtubule-associated protein, providing insights into the mechanism of protein navigation through a complex microtubule network.

Visualizing Single-Cell Secretion Dynamics with Single-Protein Sensitivity.

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein-Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa-1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity.

Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites.

Detection of single analyte molecules without the use of any label would improve the sensitivity of current biosensors by orders of magnitude to the ultimate graininess of biological matter. Over two decades, scientists have succeeded in pushing the limits of optical detection to single molecules using fluorescence. However, restrictions in photophysics and labelling protocols make this technique less attractive for biosensing. Recently, mechanisms based on vibrational spectroscopy, photothermal detection, plasmonics and microcavities have been explored for fluorescence-free detection of single biomolecules. Here, we show that interferometric detection of scattering (iSCAT) can achieve this goal in a direct and label-free fashion. In particular, we demonstrate detection of cancer marker proteins in buffer solution and in the presence of other abundant proteins. Furthermore, we present super-resolution imaging of protein binding with nanometer localization precision. The ease of iSCAT instrumentation promises a breakthrough for label-free studies of interactions involving proteins and other small biomolecules.

High-resolution biosensor based on localized surface plasmons.

We report on a new biosensor with localized surface plasmons (LSP) based on an array of gold nanorods and the total internal reflection imaging in polarization contrast. The sensitivity of the new biosensor is characterized and a model detection of DNA hybridization is carried out. The results are compared with a reference experiment using a conventional high-resolution surface plasmon resonance (SPR) biosensor. We show that the LSP-based biosensor delivers the same performance as the SPR system while involving significantly lower surface densities of interacting molecules. We demonstrate a limit of detection of 100 pM and a surface density resolution of only 35 fg×mm-2 that corresponds to less than one DNA molecule per nanoparticle on average.

Local refractive index sensitivity of plasmonic nanoparticles.

We report on an experimental characterization of the sensitivity of localized surface plasmons (LSP) to local changes in the refractive index at a nanometer scale. The method is based on forming a polymer mask covering different well defined areas of metallic nanoparticles and measuring the extinction peak shifts associated with the local refractive index changes. Arrays of nanoparticles (nanorod chains) are prepared using electron beam lithography and the dielectric mask is aligned with respect to the nanoparticle array in a second lithographic step. Extinction peak shifts corresponding to different positions of the mask are measured and values for the local refractive index sensitivity are deduced. A deconvolution procedure is established and used to map the local sensitivity across the surface of nanoparticle based on measured data. The experimental results are shown to correspond well with theoretical simulations obtained using the finite-difference time-domain method. The results indicate that the sensitivity is strongly correlated with the profile of the LSP electric field.

Real-time monitoring of biomolecular interactions in blood plasma using a surface plasmon resonance biosensor.

We report a novel approach to biosensor-based observations of biomolecular interactions which enables real-time monitoring of biomolecular interactions in complex media. This approach is demonstrated by investigating the interaction between the human chorionic gonadotropin (hCG) and its antibody in blood plasma using a surface plasmon resonance biosensor and a dispersionless microfluidics system. The real-time binding data obtained in blood plasma are compared with those obtained in buffer and blood plasma using a conventional method. It is also demonstrated that the proposed approach can enhance the capability of the biosensor to detect biomolecules in complex samples in terms of detection time and sensitivity. In the model experiment, this approach is shown to enable direct detection of hCG in blood plasma at levels which are five times lower than those detected using the conventional detection approach.

Surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma.

Surface plasmon resonance (SPR) biosensor for high-throughput screening of protein biomarkers in diluted blood plasma is reported. The biosensor combines a high-resolution SPR imaging sensor and a high-density protein array with low-fouling background. The SPR imaging sensor utilizes polarization contrast and advanced referencing and provides a total of 120 sensing areas (each 200 µm×150 µm). Antibodies are immobilized on the sensing areas via hybridization of antibody-oligonucleotide conjugates to thiolated complementary oligonucleotides microspotted on the sensor surface (DNA-directed immobilization). A low-fouling background is achieved by covalent immobilization of bovine serum albumin to carboxyl-terminated thiols filling the areas among the thiolated oligonucleotides and outside the sensing areas. The biosensor was evaluated for detection of protein biomarkers relevant to cancer diagnostics--human chorionic gonadotropin (hCG) and activated leukocyte cell adhesion molecule (ALCAM) both in buffer and in 10% blood plasma. Limits of detection as low as 45 ng/mL (ALCAM) and 100 ng/mL (hCG) were achieved in blood plasma samples.

A label-free and portable multichannel surface plasmon resonance immunosensor for on site analysis of antibiotics in milk samples.

Techniques for immunosensing like surface plasmon resonance (SPR) may respond to the need for rapid screening methods to improve food safety. This paper describes the development of a novel portable six channel SPR biosensor based on the plasmon of gold diffraction grating surface for simultaneous multianalyte antibiotic detection in milk samples. Representative congeners from three important antibiotic families (FQs: fluoroquinolones, SAs: sulfonamides and CAP: phenicols) were chosen for this study. The chips are covalently biofunctionalized with haptenized proteins by means of a previously formed mixed self assembled monolayer (m-SAM) prepared using two types of mercapto alkyl reagents containing polyethyleneglycol (PEG) units. The samples or standards are mixed with specific polyclonal antibodies and injected into the sensor device. The detectability accomplished is very good (i.e. in buffer, enrofloxacin, 0.30 µg L(-1); sulfapyridine, 0.29 µg L(-1); and chloramphenicol, 0.26 µg L(-1)) and whole milk samples can be analyzed directly without clean-up steps, by just diluting the sample five times with water to remove non-specific interferences caused by the matrix. Although the detectability of CAP regarding the MRPL (minimum required performance limit) is slightly compromised by the dilution, the detectability accomplished by FQs and SAs was far below the maximum residue levels (MRLs) established by the European Union.

Ultra-low fouling and functionalizable zwitterionic coatings grafted onto SiO2 via a biomimetic adhesive group for sensing and detection in complex media.

Non-specific protein binding from human plasma and serum has severely hindered the full capabilities of biosensors concerned with cancer biomarker detection. Currently, there is a strong desire for developing new materials which allow for the convenient attachment of an ultra-low fouling and functionalizable surface coating which can be used for highly sensitive and label-free detection of target analytes directly from complex media. In this work, a short 20 min in situ "graft to" protocol using Tris pH 8.5 buffer was developed for zwitterionic carboxybetaine methacrylate (CBMA) polymer conjugates containing the adhesive biomimetic moiety, 3,4-dihydroxy-L-phenylalanine (DOPA), on SiO(2) substrates. Using a surface plasmon resonance (SPR) biosensor, different buffers, pH values, salt concentrations, and temperatures were investigated for determining the "graft to" conditions that yield dense polymer films which both minimize non-specific protein adsorption and maximize antibody immobilization. The optimized surface coatings were shown to be highly protein resistant to 100% human blood plasma and serum. Subsequent antibody functionalized surfaces without any blocking agents enabled the specific detection of the cancer biomarker ALCAM directly from undiluted human serum down to 64 ng/mL. The successful use of this zwitterionic surface coating for detection from complex media on SiO(2) surfaces indicates its potential for broad impacts in the development of implantable medical devices, in vivo diagnostics, and nano-scale biosensors.

Surface plasmon resonance (SPR) sensors: approaching their limits?

We report on a unified theoretical model of the resolution of SPR sensors which makes it possible to predict the ultimate performance of all major configurations of SPR sensors. The theory indicates that the performance of SPR sensors is independent of the method of excitation of surface plasmons (prism or grating coupling) or the method of modulation (amplitude, angular or wavelength) and depends dominantly on the noise properties of the light source and detector. Results of the theoretical analysis are compared with the performance reported for several SPR sensors to illustrate that the best state-of-art SPR sensors are approaching their theoretical limits. Possibilities for further advances in the performance of SPR sensor technology are discussed.

Surface plasmon resonance biosensing.

Surface plasmon resonance (SPR) biosensors belong to label-free optical biosensing technologies. The SPR method is based on optical measurement of refractive index changes associated with the binding of analyte molecules in a sample to biorecognize molecules immobilized on the SPR sensor. Since late 1990's, SPR biosensors have become the main tool for the study of biomolecular interactions both in life science and pharmaceutical research. In addition, they have been increasingly applied in the detection of chemical and biological substances in important areas such as medical diagnostics, environmental monitoring, food safety and security. This chapter reviews the main principles of SPR biosensor technology and discusses applications of this technology for rapid, sensitive and specific detection of chemical and biological analytes.

Label-free detection of cancer biomarker candidates using surface plasmon resonance imaging.

In this work, we present an antibody array for the detection of cancer biomarker candidates by a surface plasmon resonance (SPR) imaging sensor with polarization contrast. Responses from the SPR imaging sensor are shown to be similar to those from a conventional spectroscopy-based SPR sensor. Antibodies are spotted onto a self-assembled monolayer (SAM) composed of oligo(ethylene glycol) (OEG)-containing alkanethiol chains. Detection of two cancer biomarker candidates, activated leukocyte cell adhesion molecule/CD 166 (ALCAM) and transgelin-2 (TAGLN2), is demonstrated. Limits of detection for ALCAM and TAGLN2 are established at 6 ng/mL and 3 ng/mL, respectively, in buffer. No cross-reactivity is observed between immobilized antibodies and nonspecific antigen. Biomarker candidates are also detected in a 10% human serum solution.

Functionalizable surface platform with reduced nonspecific protein adsorption from full blood plasma--material selection and protein immobilization optimization.

In this work, zwitterionic polymers are investigated as ultra-low fouling and functionalizable coatings for biosensors, nanoparticle-based diagnostics, and microarrays to enable detections in real-world complex media. The effect of the spacer length between the two charged groups on the nonfouling properties of zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) was studied in blood plasma and serum. The polyCBAA polymer with an ethylene spacer was selected for protein immobilization studies. A polyCBAA-coated surface was functionalized with antibodies using a simple and fast amino coupling chemistry for direct protein immobilization in two simple steps: surface activation and protein immobilization/background deactivation. The effect of pH was found to be very important for both steps and it was optimized. The functionalized polyCBAA surface exhibited very low fouling properties even when exposed to undiluted blood plasma for more than 6h with <7ng/cm(2) of adsorbed proteins. The biological activity of the immobilized proteins was demonstrated with the detection of a model protein in undiluted blood plasma. A recently developed highly sensitive four-channel surface plasmon resonance (SPR) sensor was used for the evaluation of specific and nonspecific protein adsorption to these surfaces.

High-throughput SPR sensor for food safety.

High-throughput surface plasmon resonance (SPR) biosensor for rapid and parallelized detection of nucleic acids identifying specific bacterial pathogens is reported. The biosensor consists of a high-performance SPR imaging sensor with polarization contrast and internal referencing (refractive index resolution 2 x 10(-7) RIU) and an array of DNA probes microspotted on the surface of the SPR sensor. It is demonstrated that short sequences of nucleic acids (20-23 bases) characteristic for bacterial pathogens such as Brucella abortus, Escherichia coli, and Staphylococcus aureus can be detected at 100 pM levels. Detection of specific DNA or RNA sequences can be performed in less than 15 min by the reported SPR sensor.

Compact and low-cost biosensor based on novel approach to spectroscopy of surface plasmons.

A high-performance surface plasmon resonance (SPR) sensor based on a novel approach to spectroscopy of surface plasmons is reported. This approach employs a special diffraction grating structure (referred to as surface plasmon resonance coupler and disperser, SPRCD) which simultaneously couples light into a surface plasmon and disperses the diffracted light for spectral readout of SPR signal. The developed SPRCD sensor consists of a miniature cartridge integrating the diffraction grating and microfluidics and a compact optical system which simultaneously acquires data from four independent sensing channels in the cartridge. It is demonstrated that the SPRCD sensor is able to measure bulk refractive index changes as small as 3 x 10(-7) RIU (refractive index units) and to detect short oligonucleotides in concentrations down to 200 pM.

Ultralow fouling and functionalizable surface chemistry based on a zwitterionic polymer enabling sensitive and specific protein detection in undiluted blood plasma.

A crucial step in the development of implanted medical devices, in vivo diagnostics, and microarrays is the effective prevention of nonspecific protein adsorption from real-world complex media such as blood plasma or serum. In this work, a zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) biomimetic material was employed to create a unique biorecognition coating with an ultralow fouling background, enabling the sensitive and specific detection of proteins in blood plasma. Conditions for surface activation, protein immobilization, and surface deactivation of the carboxylate groups in the polyCBAA coating were determined. An antibody-functionalized polyCBAA surface platform was used to detect a target protein in blood plasma using a sensitive surface plasmon resonance (SPR) sensor. A selective protein was directly detected from 100% human blood plasma with extraordinary specificity and sensitivity. The total nonspecific protein adsorption on the functionalized polyCBAA surface was very low (<3 ng/cm (2) for undiluted blood plasma). Because of the significant reduction of nonspecific protein adsorption, it was possible to monitor the kinetics of antigen-antibody interactions in undiluted blood plasma. The functionalization effectiveness and detection characteristics using a cancer protein marker candidate of polyCBAA were compared with those of the conventional nonfouling oligo(ethylene glycol)-based surface chemistry.