Label-free Molecule Detection Research Articles

Antibody binding to plasmonic nanostructure: A validation study using experiment and simulation

Localized surface plasmon resonance (LSPR) sensors allow the label-free detection of molecules in real-time with a high sensitivity, which makes them suitable for the detection of various molecules. However, the experimental characterization of the surface functionalization and the molecular binding on the nanostructured sensor surface represent black boxes. To overcome these uncertainties, this study shows an approach to simulate and experimentally validate the plasmonic response upon molecular binding to a LSPR sensor for the detection of diclofenac (DCF). Assuming homogeneous molecular coverage and averaged parameters such as refractive index and layer thickness through the combination of an iterative experimental validation and electrodynamics simulations a coherent model was formed that includes the uncertainties of molecular binding. This validated model and the obtained parameter for the biochemical assay forms the basis for a future automated optimization of the nanostructure geometry with respect to increased sensitivity to molecular binding.

Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections.

A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can localize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study, we use a new data-acquisition scheme called Defocus-Corrected Large-Area cryo-EM (DeCo-LACE) to collect high-resolution images of entire sections (100- to 250-nm-thick lamellae) of neutrophil-like mouse cells, representing 1-2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide maps of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods.

Single-molecule optofluidic microsensor with interface whispering gallery modes.

Label-free sensors are highly desirable for biological analysis and early-stage disease diagnosis. Optical evanescent sensors have shown extraordinary ability in label-free detection, but their potentials have not been fully exploited because of the weak evanescent field tails at the sensing surfaces. Here, we report an ultrasensitive optofluidic biosensor with interface whispering gallery modes in a microbubble cavity. The interface modes feature both the peak of electromagnetic-field intensity at the sensing surface and high-Q factors even in a small-sized cavity, enabling a detection limit as low as 0.3 pg/cm2 The sample consumption can be pushed down to 10 pL due to the intrinsically integrated microfluidic channel. Furthermore, detection of single DNA with 8 kDa molecular weight is realized by the plasmonic-enhanced interface mode.

Integration of sequential analytical processes into sub-100 nm channels: volumetric sampling, chromatographic separation, and label-free molecule detection.

The progress of nanotechnology has developed nanofluidic devices utilizing nanochannels with a width and/or depth of sub-100 nm (101 nm channels), and several experiments have been implemented in ultra-small spaces comparable to DNAs and proteins. However, current experiments utilizing 101 nm channels focus on a single function or operation; integration of multiple analytical operations into 101 nm channels using nanofluidic circuits and fluidic control has yet to be realized despite the advantage of nanochannels. Herein, we report the establishment of a label-free molecule detection method for 101 nm channels and demonstration of sequential analytical processes using integrated nanofluidic devices. Our absorption-based detection method called photothermal optical diffraction (POD) enables non-invasive label-free molecule detection in 101 nm channels for the first time, and the limit of detection (LOD) of 1.8 μM is achieved in 70 nm wide and deep nanochannels, which corresponds to 7.5 molecules in the detection volume of 7 aL. As a demonstration of sampling in 101 nm channels, aL-fL volumetric sampling is performed using 90 nm deep cross-shaped nanochannels and pressure-driven fluidic control from three directions. Finally, the POD and volumetric sampling are combined with nanochannel chromatography, and separation analysis in 101 nm channels is demonstrated. The experimental results reported in this paper will contribute to the advances in 101 nm fluidic devices which have the potential to provide a novel platform for chemical/biological analyses.

Nonlinear Sensing with Whispering-Gallery Mode Microcavities: From Label-Free Detection to Spectral Fingerprinting.

Optical microresonators have attracted intense interests in highly sensitive molecular detection and optical precision measurement in the past decades. In particular, the combination of a high quality factor with a small mode volume significantly enhances the nonlinear light-matter interaction in whispering-gallery mode (WGM) microresonators, which greatly boost nonlinear optical sensing applications. Nonlinear WGM microsensors not only allow for label-free detection of molecules with an ultrahigh sensitivity but also support new functionalities in sensing such as the specific spectral fingerprinting of molecules with frequency conversion involved. Here, we review the mechanisms, sensing modalities, and recent progresses of nonlinear optical sensors along with a brief outlook on the possible future research directions of this rapidly advancing field.

Nanochannel chromatography and photothermal optical diffraction: Femtoliter sample separation and label-free zeptomole detection

Column miniaturization of liquid chromatography is a major trend in separation sciences with the advent of single-cell proteomics and metabolomics. Nanochannel chromatography is one of the promising tools for single-cell analyses because it provides ultra-small sample volume and high separation efficiency. However, non-fluorescent molecular detection in such small channels is still quite difficult due to fL–aL sample volume, which hinders further miniaturization of nanochannels. In this study, we overcame the size limitation of nanochannel chromatography by our label-free molecular detection method: photothermal optical diffraction (POD), which utilizes the photothermal effect of analytes and optical diffraction by nanochannel. The combination of the nanochannel chromatography and the POD enables 1.8 fL sample separation and label-free molecule detection in nanochannels with 800 nm width and 300 nm depth at the optimized experimental conditions. The limit of detection is 5.4 zmol (3300 molecules), approximately 50 times lower than the conventional label-free detection method. Furthermore, the theoretical plate number is calculated to be 105 plates/m, and the separation performance is discussed. Our label-free detection method will be widely used as a universal detector for nanochannel chromatography.

Photonic Crystal Nanobeam Cavities for Nanoscale Optical Sensing: A Review

The ability to detect nanoscale objects is particular crucial for a wide range of applications, such as environmental protection, early-stage disease diagnosis and drug discovery. Photonic crystal nanobeam cavity (PCNC) sensors have attracted great attention due to high-quality factors and small-mode volumes (Q/V) and good on-chip integrability with optical waveguides/circuits. In this review, we focus on nanoscale optical sensing based on PCNC sensors, including ultrahigh figure of merit (FOM) sensing, single nanoparticle trapping, label-free molecule detection and an integrated sensor array for multiplexed sensing. We believe that the PCNC sensors featuring ultracompact footprint, high monolithic integration capability, fast response and ultrahigh sensitivity sensing ability, etc., will provide a promising platform for further developing lab-on-a-chip devices for biosensing and other functionalities.

Theoretical analysis of bimetallic nanorod dimer biosensors for label-free molecule detection

In this work, we theoretically analyze a gold (Au) core within silver (Ag) shell (Au@Ag) nanorod dimer biosensor for label-free molecule detection. The incident light on an Au@Ag nanorod strongly couples to localized surface plasmon modes, especially around the tip region. The field enhancement around the tip of a nanorod or between the tips of two longitudinally aligned nanorods as in a dimer can be exploited for sensitive detection of biomolecules. We derive analytical expressions for the interactions of an Au@Ag nanorod dimer with the incident light. We also study the detail dynamics of an Au@Ag nanorod dimer with the incident light computationally using finite difference time domain (FDTD) technique when core-shell ratio, relative position of the nanorods, and angle of incidence of light change. We find that the results obtained using the developed analytical model match well with that obtained using FDTD simulations. Additionally, we investigate the sensitivity of the Au@Ag nanorod dimer, i.e., shift in the resonance wavelength, when a target biomolecule such as lysozyme (Lys), human serum albumin (HSA), anti-biotin (Abn), human catalase (CAT), and human fibrinogen (Fb) protein molecules are attached to the tips of the nanorods.

A differential weak measurement system based on Sagnac interferometer for self-referencing biomolecule detection

A differential weak measurement system was presented, exhibiting the self-referencing function for biomolecule real time detection as a label-free optical biosensor. We built a Sagnac interferometer, which limited horizontal (H) and vertical (V) polarization to propagating along the common path but in opposite directions to realize weak measurements with two measuring channels installed in two corners of this Sagnac interferometer. By introducing two half wave plates into the system alternately with the two channels to convert between H and V polarization, we obtained a differential measurement for phase delay, which could quantitatively characterize the refractive index change corresponding to the concentration of samples in the channels. With this system, a real time monitor of molecule concentration in the dialysis process was accomplished, demonstrating the function of self-referencing, which is important for optical label-free molecule detection in a complex biological sample solution.

Preparation and detection of progesterone by molecularly imprinted film based on reversible addition fragmentation chain transfer polymerization

Molecularly imprinted technique is a versatile and straightforward way to produce artificial receptors for a given target molecule. The molecularly imprinted polymers (MIPs) have received widespread attention and application, owing to their high physical stability, remarkable robustness and low cost. Despite the tremendous progress made in this field, many changes still remain to be solved. In general, MIPs are prepared by traditional free radical polymerization. However, this approach causes problems such as low-affinity binding, high diffusion barrier, low-rate mass transfer. In particular, it has been demonstrated that most of the MIPs fail to exhibit specific recognition towards the target molecules in aqueous environments although they can be successfully applied to recognize the target molecules in organic solvent media, which severely limits their potential application in some fields. In this work, a new method to prepare MIPs film for the detection of progesterone based on reversible addition-fragmentation chain transfer (RAFT) polymerization is presented. 2-Methyl-2-[(dodecylsulfanylthiocarbonyl)sulfanyl]propanoic acid (DDMAT) was employed as the sole control agent in the UV polymerization. The SPR sensor chip with 50 nm Au was firstly immersed in an ethanol solution of cysteamine (5 mmol/L) for 24 h. The cysteamine modificated chip was washed by pure ethanol solvent repeatedly and dried under a stream of nitrogen. Then the chip was placed in 40 mL acetonitrile solution with 14.6 mg DDMAT and 16.5 mg N , N ′-dicyclohexylcarbodiimide (DCC) for 24 h. After that, the chip was washed with acetonitrile several times and dried under a stream of nitrogen. The MIPs film was grafted by UV irradiated without adding additional photo initiator or catalyst in the presence of progesterone (31.4 mg), methacrylic acid (33.4 μL), ethylene glycol dimethacrylate (338.5μL) and DDMAT (15 mg) in 4 mL acetonitrile. The growth process of the MIPs film was monitored by surface plasmon resonance (SPR) in situ . A mixture of PBS (pH 7.4) buffer and acetic acid (v:v=9:1) was used as a washing solution to remove the template molecules from the MIPs film. Results indicated that DDMAT acted as both photo initiator and chain transfer agent during the polymerization. The MIPs film was characterized by contact angle measurements, infrared spectroscopy and scanning electron microscope (SEM). Analysis of SPR spectroscopy showed that the MIPs film coated sensor displayed good sensitivity and selectivity to progesterone compared to other analytes with a similar structure. The response of the progesterone ranged from 10−12 to 10−7 mol/L with a limit of detection (LOD) of 3.24×10−13 mol/L ( S / N =3) in PBS buffer (pH 7.4). The MIPs film coated sensor also showed good reusability and stability, and kept about 81% of its original response after 30 d of storage under N2 protection at room temperature. In addition, the SPR sensor was successfully used to determine progesterone in tap water, underground water and artificial urine samples with recoveries from 92.9% to 96.5%, which indicated the SPR sensor can be applied for the real sample detection. This method will expand the surface modification of the MIPs film on the surface of different sensors and is a great candidate for rapid, simple and label-free detection of molecules in aqueous solution. Finally, this study opens a door to re-know the roles of chain transfer agents (DDMAT, in this study) in synthesizing MIPs and we believe that the properties of various chain transfer agents will be excavated and utilized in future study.

Silver Alginate Hydrogel Micro- and Nanocontainers for Theranostics: Synthesis, Encapsulation, Remote Release, and Detection.

We have designed multifunctional silver alginate hydrogel microcontainers referred to as loaded microcapsules with different sizes by assembling them via a template assisted approach using natural, highly porous calcium carbonate cores. Sodium alginate was immobilized into the pores of calcium carbonate particles of different sizes followed by cross-linking via addition of silver ions, which had a dual purpose: on one hand, the were used as a cross-linking agent, albeit in the monovalent form, while on the other hand they have led to formation of silver nanoparticles. Monovalent silver ions, an unusual cross-linking agent, improve the sensitivity to ultrasound, lead to homogeneous distribution of silver nanoparticles. Silver nanoparticles appeared on the shell of the alginate microcapsules in the twin-structure as determined by transmission electron microscopy. Remote release of a payload from alginate containers by ultrasound was found to strongly depend on the particle size. The possibility to use such particles as a platform for label-free molecule detection based on the surface enhanced Raman scattering was demonstrated. Cytotoxicity and cell uptake studies conducted in this work have revealed that microcontainers exhibit nonessential level of toxicity with an efficient uptake of cells. The above-described functionalities constitute building blocks of a theranostic system, where detection and remote release can be achieved with the same carrier.

Terahertz probe of individual subwavelength objects in a water environment

Terahertz (THz) spectroscopy and imaging have been heralded for some time as potentially revolutionary techniques for biomedical applications. Label-free detection of molecules and recognition of molecular events are often mentioned as the most exciting possibilities. A crucial practical goal, however, is the ability to perform such measurements on tiny amounts of biological fluids or even on individual organic structures. Living cells, for instance, have diameters at most of some tens of micrometers, i.e. at least λ/10 even for few-THz radiation. Furthermore, all analyses relevant for a biological perspective must be performed in a water environment, which presents a strong absorption across the whole THz spectral range, severely limiting the penetration of the electromagnetic field. Here, it is shown how both issues can be overcome with a lab-on-a-chip approach based on a microfluidic platform coupled to a plasmonic antenna. Using a quantum cascade laser as THz illumination source, liquid volumes down to the picoliter range are probed, and direct operation on individual 10-µm diameter microparticles flowing in water is shown. The present demonstration opens the way to the development of THz biosensing of individual living cells and small probe volumes.

Optical sensing of square lattice photonic crystal point-shifted nanocavity for protein adsorption detection

We propose a point-shifted D0 nanocavity formed by locally modulating four central air holes in square lattice photonic crystal for optical sensing application. Three defect modes in this nanocavity, including monopole, whispering-gallery, and dipole modes, are identified in experiments. We also apply a chemical treatment on InGaAsP surface to form a 1-octadecanethiol linking monolayer, which enables the following protein adsorption. In experiments, the wavelength shifts of lasing modes in the D0 nanocavity due to the protein adsorption are observed and agree with the simulation results. This can be a practical tool for label-free molecule detection in biomedical researches.

Autofluorescence Detection in Analytical Chemistry and Biochemistry

Label-free detection of molecules and particles based on native fluorescence excited at the ultraviolet (UV) region of the electromagnetic spectrum shows great potential for molecular analysis in life sciences. It offers simple, low-cost, and fast methods for sensitive detection of important biological analytes in nature forms. This article reviews the most significant research on label-free native fluorescence detection in analytical chemistry and biochemistry, with particular focus on the instrumental requirements and applications that cover papers published to the beginning of 2009. Future development directions for label-free optical biosensors based on native fluorescence are discussed.

Microfabricated polymer chip with integrated U-bend waveguides for evanescent field absorption based detection

A mu-TAS system for evanescent field absorption with integrated polymer waveguides is reported for the first time. A photoresist SU-8 layer is patterned into a microchannel network, with U-bend waveguides and fiber-to-waveguide coupler structures. The aim of this study was to explore the possibility of using evanescent field absorption based sensing in conjunction with capillary electrophoresis for label free detection. We have proposed a novel design to couple the microchannel network with U-bend waveguides in a single step patterning of SU-8. In this novel design, the optical waveguide forms part of the microchannel wall, which aids in the detection process. The suitability of the device for optical applications was proved by absorbance measurement between 450 and 780 nm using Methylene Blue dye. Absorbance measurements were done by passing various concentrations of dye solutions through 200 microm and 500 microm microchannels. The device was also found sensitive to the refractive index (RI) of fluid flowing in the channel. The RI sensitivity was tested by passing sucrose solutions of varying concentrations through the channels and measuring absorbance across the integrated U-bend waveguides. The results indicate that such structures can be used easily for label free detection of molecules either by evanescent wave absorption or by changes associated with RI changes in the microenvironment around a waveguide.

Label-free optical biosensing with slot-waveguides

We demonstrate label-free molecule detection by using an integrated biosensor based on a Si(3)N(4)/SiO(2) slot-waveguide microring resonator. Bovine serum albumin (BSA) and anti-BSA molecular binding events on the sensor surface are monitored through the measurement of resonant wavelength shifts with varying biomolecule concentrations. The biosensor exhibited sensitivities of 1.8 and 3.2 nm/(ng/mm(2)) for the detection of anti-BSA and BSA, respectively. The estimated detection limits are 28 and 16 pg/mm(2) for anti-BSA and BSA, respectively, limited by wavelength resolution.

Quartz Crystal Microbalance for Bioanalytical Applications

AbstractFor Abstract see ChemInform Abstract in Full Text.

Quartz Crystal Microbalance for Bioanalytical Applications

The quartz crystal microbalance (QCM) was first introduced as a mass sensor in gas phase and in vacuum. Since oscillator circuits capable of exciting shear vibrations of quartz resonators under liquid load have been developed, the QCM became accepted as a new powerful technique to monitor adsorption processes at solid/liquid interfaces in chemical and biological research rendering the method an attractive low-cost alternative for bioanalytic applications. In the last decade, adsorption of biomolecules on functionalized surfaces turned out to be one of the paramount applications of piezoelectric transducers comprising the interaction of DNA and RNA with complementary strands, specific recognition of protein-ligands by immobilized receptors, the detection of virus capsids, bacteria, mammalian cells and last but not least the development of complete immunosensors. Piezoelectric transducers allow a label-free detection of molecules; they are more than mere mass sensors since the sensor response is also influenced by interfacial phenomena, viscoelastic properties of the adhered biomaterial, surface charges of adsorbed molecules and surface roughness. These new insights have recently been used to investigate the adhesion of cells, liposomes and proteins onto surfaces allowing to determine morphological changes of cells as a response to pharmacological substances and changes in the water content of biopolymers in situ. However, future will show whether the quartz crystal microbalance will assert itself against established label-free sensor devices like surface plasmon resonance spectroscopy and interferometry.

Piezoelectric Mass-Sensing Devices as Biosensors-An Alternative to Optical Biosensors?

In the early days of electronic communication-as a result of the limited number of quartz resonators available-frequency adjustment was accomplished by a pencil mark depositing a foreign mass layer on the crystal. In 1959, Sauerbrey showed that the shift in resonance frequency of thickness-shear-mode resonators is proportional to the deposited mass. This was the starting point for the development of a new generation of piezoelectric mass-sensitive devices. However, it was the development of new powerful oscillator circuits that were capable of operating thickness shear mode resonators in fluids that enabled this technique to be introduced into bioanalytic applications. In the last decade adsorption of biomolecules on functionalized surfaces turned in to one of the paramount applications of piezoelectric transducers. These applications include the study of the interaction of DNA and RNA with complementary strands, specific recognition of protein ligands by immobilized receptors, the detection of virus capsids, bacteria, mammalian cells, and last but not least the development of complete immunosensors. Piezoelectric transducers allow a label-free detection of molecules; they are more than mere mass sensors since the sensor response is also influenced by interfacial phenomena, viscoelastic properties of the adhered biomaterial, surface charges of adsorbed molecules, and surface roughness. These new insights have recently been used to investigate the adhesion of cells, liposomes, and proteins onto surfaces, thus allowing the determination of the morphological changes of cells as a response to pharmacological substances and changes in the water content of biopolymers without employing labor-intense techniques. However, the future will show whether the quartz-crystal microbalance will assert itself against established label-free sensor devices such as surface plasmon resonance spectroscopy and interferometry.