Label-free Measurement Research Articles

Non-invasive Measurement of Brown Fat Metabolism Based on Optoacoustic Imaging of Hemoglobin Gradients

Metabolism is a fundamental process of life. However, non-invasive measurement of local tissue metabolism is limited today by a deficiency in adequate tools for invivo observations. We designed a multi-modular platform that explored the relation between local tissue oxygen consumption, determined by label-free optoacoustic measurements of hemoglobin, and concurrent indirect calorimetry obtained during metabolic activation of brown adipose tissue (BAT). By studying mice and humans, we show how video-rate handheld multi-spectral optoacoustic tomography (MSOT) in the 700-970nm spectral range enables non-invasive imaging of BAT activation, consistent with positron emission tomography findings. Moreover, we observe BAT composition differences between healthy and diabetic tissues. The study consolidates hemoglobin as a principal label-free biomarker for longitudinal non-invasive imaging of BAT morphology and bioenergetics insitu. We also resolve water and fat components in volunteers, and contrast MSOT readouts with magnetic resonance imaging data.

Multiplex Surface Plasmon Resonance Imaging-Based Biosensor for Human Pancreatic Islets Hormones Quantification.

Diabetes arises from secretory defects in vascularized micro-organs known as the islets of Langerhans. Recent studies indicated that furthering our understanding of the paracrine effect of somatostatin on glucose-induced insulin secretion could represent a novel therapeutic avenue for diabetes. While many research groups are interested in insulin and glucagon secretion, few are particularly focused on studying the paracrine interaction in islets' cells, and none on monitoring a secretory fingerprint that contemplates more than two hormones. Surface plasmon resonance imaging can achieve high-throughput and multiplexed biomolecule quantification, making it an ideal candidate for detection of multiple islet's secretion products if arrays of hormones can be properly implemented on the sensing surface. In this study, we introduced a multiplex surface plasmon resonance imaging-based biosensor for simultaneous quantification of insulin, glucagon, and somatostatin. Performing this multiplex biosensing of hormones was mainly the result of the design of an antifouling sensing surface comprised by a mixed self-assembly monolayer of CH3O-PEG-SH and 16-mercaptohexadecanoic acid, which allowed it to operate in a complex matrix such as an islet secretome. The limit of detection in multiplex mode was 1 nM for insulin, 4 nM for glucagon, and 246 nM for somatostatin with a total analysis time of 21 min per point, making our approach the first reporting a label-free and multiplex measurement of such a combination of human hormones. This biosensor holds the promise of providing us with a mean for the further understanding of the paracrine effect of somatostatin on glucose-induced insulin secretion and consequently help develop novel therapeutic agents for diabetes.

Surface-Enhanced Raman Spectroscopy-Based Label-Free Insulin Detection at Physiological Concentrations for Analysis of Islet Performance.

Label-free optical detection of insulin would allow in vitro assessment of pancreatic cell functions in their natural state and expedite diabetes-related clinical research and treatment; however, no existing method has met these criteria at physiological concentrations. Using spatially uniform 3D gold-nanoparticle sensors, we have demonstrated surface-enhanced Raman sensing of insulin in the secretions from human pancreatic islets under low and high glucose environments without the use of labels such as antibodies or aptamers. Label-free measurements of the islet secretions showed excellent correlation among the ambient glucose levels, secreted insulin concentrations, and measured Raman-emission intensities. When excited at 785 nm, plasmonic hotspots of the densely arranged 3D gold-nanoparticle pillars as well as strong interaction between sulfide linkages of the insulin molecules and the gold nanoparticles produced highly sensitive and reliable insulin measurements down to 100 pM. The sensors exhibited a dynamic range of 100 pM to 50 nM with an estimated detection limit of 35 pM, which covers the reported concentration range of insulin observed in pancreatic cell secretions. The sensitivity of this approach is approximately 4 orders of magnitude greater than previously reported results using label-free optical approaches, and it is much more cost-effective than immunoassay-based insulin detection widely used in clinics and laboratories. These promising results may open up new opportunities for insulin sensing in research and clinical applications.

Multiparameter cell-tracking intrinsic cytometry for single-cell characterization.

An abundance of label-free microfluidic techniques for measuring cell intrinsic markers exists, yet these techniques are seldom combined because of integration complexity such as restricted physical space and incompatible modes of operation. We introduce a multiparameter intrinsic cytometry approach for the characterization of single cells that combines ≥2 label-free measurement techniques onto the same platform and uses cell tracking to associate the measured properties to cells. Our proof-of-concept implementation can measure up to five intrinsic properties including size, deformability, and polarizability at three frequencies. Each measurement module along with the integrated platform were validated and evaluated in the context of chemically induced changes in the actin cytoskeleton of cells. viSNE and machine learning classification were used to determine the orthogonality between and the contribution of the measured intrinsic markers for cell classification.

Fiber interferometer for hybrid optical and optoacoustic intravital microscopy

The addition of optoacoustic sensing to optical microscopy may supplement fluorescence contrast with label-free measurements of optical absorption, enhancing biological observation. However, the physical dimensions of many optoacoustic systems have restricted the implementation of hybrid optical and optoacoustic (O2A) microscopy to imaging thin samples in transmission mode or to ex-vivo investigations. Here we describe a miniaturized optoacoustic sensor, based on a π-phase-shifted fiber Bragg grating embedded in an acoustic cavity, which is virtually invisible to the optical path and can be seamlessly integrated into any conventional optical microscope. The new sensor enables, for the first time to our knowledge, entirely optical O2A intravital microscopy in epi-illumination mode, demonstrated by label-free optoacoustic and second-harmonic generation images of a mouse abdomen and ear. Our technique greatly simplifies the integration of acoustic detection in standard microscopes and could therefore make optoacoustic microscopy more accessible to the biomedical community.

Mg2+-induced DNA compaction, condensation, and phase separation in gene delivery vehicles based on zwitterionic phospholipids: a dynamic light scattering and surface-enhanced Raman spectroscopic study.

Despite the significant efforts towards applying improved non-destructive and label-free measurements of biomolecular structures of lipid-based gene delivery vectors, little is achieved in terms of their structural relevance in gene transfections. Better understanding of structure-activity relationships of lipid-DNA complexes and their gene expression efficiencies thus becomes an essential issue. Raman scattering offers a complimentary measurement technique for following the structural transitions of both DNA and lipid vesicles employed for their transfer. This work describes the use of SERS coupled with light scattering approaches for deciphering the bioelectrochemical phase formations between nucleic acids and lipid vesicles within lipoplexes and their surface parameters that could influence both the uptake of non-viral gene carriers and the endocytic routes of interacting cells. As promising non-viral alternatives of currently employed risky viral systems or highly cytotoxic cationic liposomes, complexations of both nucleic acids and zwitterionic lipids in the presence of Mg2+ were studied applying colloidal Ag nanoparticles. It is shown that the results could be employed in further conformational characterizations of similar polyelectrolyte gene delivery systems.

Integrating Next-Generation Genomic Sequencing and Mass Spectrometry To Estimate Allele-Specific Protein Abundance in Human Brain.

Gene expression contributes to phenotypic traits and human disease. To date, comparatively less is known about regulators of protein abundance, which is also under genetic control and likely influences clinical phenotypes. However, identifying and quantifying allele-specific protein abundance by bottom-up proteomics is challenging since single nucleotide variants (SNVs) that alter protein sequence are not considered in standard human protein databases. To address this, we developed the GenPro software and used it to create personalized protein databases (PPDs) to identify single amino acid variants (SAAVs) at the protein level from whole exome sequencing. In silico assessment of PPDs generated by GenPro revealed only a 1% increase in tryptic search space compared to a direct translation of all human transcripts and an equivalent search space compared to the UniProtKB reference database. To identify a large unbiased number of SAAV peptides, we performed high-resolution mass spectrometry-based proteomics for two human post-mortem brain samples and searched the collected MS/MS spectra against their respective PPD. We found an average of ∼117 000 unique peptides mapping to ∼9300 protein groups for each sample, and of these, 977 were unique variant peptides. We found that over 400 reference and SAAV peptide pairs were, on average, equally abundant in human brain by label-free ion intensity measurements and confirmed the absolute levels of three reference and SAAV peptide pairs using heavy labeled peptides standards coupled with parallel reaction monitoring (PRM). Our results highlight the utility of integrating genomic and proteomic sequencing data to identify sample-specific SAAV peptides and support the hypothesis that most alleles are equally expressed in human brain.

Interferogram Average over Wavelength Spectroscopy: An Ultrasensitive Technique for Biosensing with Porous Silicon Interferometers

Here we report on a high-sensitivity interferometric technique, namely Interferogram Averaged over Wavelength – IAW – reflectance spectroscopy, enabling nanomolar detection (both nonspecific and specific) of proteins to be accomplished in direct (without any amplification) label-free mode using porous silicon (PSi) interferometers. The IAW reflectance spectroscopy is based on both calculation and elaboration of spectral interferograms. Interferograms are obtained by subtracting (intensity, wavelength by wavelength) reflectance spectra acquired on PSi interferometers in buffer solution after protein injection (at different concentrations) from a reference reflectance spectrum acquired in buffer solution before protein injection. The output signal, namely IAW value, is eventually obtained as the average value, calculated over a given wavelength interval, of each interferogram. Using the IAW technique nonspecific adsorption of Bovine Serum Albumin (BSA) down to 0.15 nM (DL=20 pM) and specific detection of Tumor Necrosis Factor (TNFα) down to 3 nM (DL=0.2 nM) was reliably achieved in direct label-free mode using PSi interferometers. Both the results represent a 10000-fold reduction in DL with respect to the current literature using PSi for direct label-free measurements.

Recent advances in surface plasmon resonance imaging: detection speed, sensitivity, and portability

Abstract Surface plasmon resonance (SPR) biosensor is a powerful tool for studying the kinetics of biomolecular interactions because they offer unique real-time and label-free measurement capabilities with high detection sensitivity. In the past two decades, SPR technology has been successfully commercialized and its performance has continuously been improved with lots of engineering efforts. In this review, we describe the recent advances in SPR technologies. The developments of SPR technologies focusing on detection speed, sensitivity, and portability are discussed in details. The incorporation of imaging techniques into SPR sensing is emphasized. In addition, our SPR imaging biosensors based on the scanning of wavelength by a solid-state tunable wavelength filter are highlighted. Finally, significant advances of the vast developments in nanotechnology-associated SPR sensing for sensitivity enhancements are also reviewed. It is hoped that this review will provide some insights for researchers who are interested in SPR sensing, and help them develop SPR sensors with better sensitivity and higher throughput.

Characterizing Esophageal Cancerous Cells at Different Stages Using the Dielectrophoretic Impedance Measurement Method in a Microchip.

Analysis of cancerous cells allows us to provide useful information for the early diagnosis of cancer and to monitor treatment progress. An approach based on electrical principles has recently become an attractive technique. This study presents a microdevice that utilizes a dielectrophoretic impedance measurement method for the identification of cancerous cells. The proposed biochip consists of circle-on-line microelectrodes that are patterned using a standard microfabrication processes. A sample of various cell concentrations was introduced in an open-top microchamber. The target cells were collectively concentrated between the microelectrodes using dielectrophoresis manipulation, and their electrical impedance properties were also measured. Different stages of human esophageal squamous cell carcinoma lines could be distinguished. This result is consistent with findings using hyperspectral imaging technology. Moreover, it was observed that the distinguishing characteristics change in response to the progression of cancer cell invasiveness by Raman spectroscopy. The device enables highly efficient cell collection and provides rapid, sensitive, and label-free electrical measurements of cancerous cells.

On chip two-photon metabolic imaging for drug toxicity testing.

We have developed a microfluidic system suitable to be incorporated with a metabolic imaging method to monitor the drug response of cells cultured on a chip. The cells were perfusion-cultured to mimic the blood flow in vivo. Label-free optical measurements and imaging of nicotinamide adenine dinucleotide and flavin adenine dinucleotide fluorescence intensity and morphological changes were evaluated non-invasively. Drug responses calculated using redox ratio imaging were compared with the drug toxicity testing results obtained with a traditional well-plate system. We found that our method can accurately monitor the cell viability and drug response and that the IC50 value obtained from imaging analysis was sensitive and comparable with a commonly used cell viability assay: MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium) assay. Our method could serve as a fast, non-invasive, and reliable way for drug screening and toxicity testing as well as enabling real-time monitoring of in vitro cultured cells.

Intravital microscopy of biosensor activities and intrinsic metabolic states.

Intravital microscopy (IVM) is an imaging tool that is capable of detecting subcellular signaling or metabolic events as they occur in tissues in the living animal. Imaging in highly scattering biological tissues, however, is challenging because of the attenuation of signal in images acquired at increasing depths. Depth-dependent signal attenuation is the major impediment to IVM, limiting the depth from which significant data can be obtained. Therefore, making quantitative measurements by IVM requires methods that use internal calibration, or alternatively, a completely different way of evaluating the signals. Here, we describe how ratiometric imaging of genetically encoded biosensor probes can be used to make quantitative measurements of changes in the activity of cell signaling pathways. Then, we describe how fluorescence lifetime imaging can be used for label-free measurements of the metabolic states of cells within the living animal.

Graphene and functionalized graphene: Extraordinary prospects for nanobiocomposite materials

Abstract Graphene research, already prized, is nonetheless challenged to provide material advantages that bring about unique and novel applications rather than simply an improved substitute to existing materials. Arguably driving significant progress are new approaches to its manufacture including combination with other atoms and nanobiocomposite materials that introduce new functional properties. Functionalized graphene can interface with biomolecules to provide new health sector benefits in the form of highly sensitive biosensors that may offer continuous label-free measurement of key bioactive cell molecules, innovative nanoparticles for tissue targeted drug delivery and scaffolds for tissue engineering with previously unachievable qualities favouring tissue integration and biocompatibility. We here describe graphene functionalization methods and provide recent examples of how graphene can be used to achieve biological interactions with innovative outcomes. Above all, these laboratory focused advances each contribute to an incrementally improved understanding of how different forms of graphene and its derivatives can best be tailored to meet biological demands. Much has yet to be discovered with regard to safety profiles and improved manufacture yet considering how carefully derived rational insights might be combined with computational biology to accelerate complex performance models, prospects that graphene-based biomaterials can achieve extraordinary real-world benefits are optimistically poised.

One-Step Ligand Immobilization and Single Sample Injection for Regeneration-Free Surface Plasmon Resonance Measurements of Biomolecular Interactions.

Surface plasmon resonance (SPR) has been well established as a method of choice for label-free kinetic measurements of biomolecular interactions. The conventional approach involves multiple injections of an analyte of different concentrations into a fluidic channel covered with a fixed ligand density. Optimization of the experimental conditions and assessment of the data quality can be complicated by issues such as disruption of the ligand structure by the regeneration step and the limited availability of the sample solution. By sequentially closing fluidic channels on a five-channel SPR instrument, different densities of a ligand can be immobilized and determined in one step. With a subsequent injection of a single sample solution, SPR sensorgrams can be simultaneously collected to yield binding and dissociation rate constants (ka and kd) and dissociation constant (KD) between the ligand and analyte. For biomolecular interactions that obey the Langmuir isotherm, we show that the fidelity of the kinetic data can only be reliably confirmed when there exists a strong linear correlation between the SPR signals and the ligand densities. The use of a multichannel SPR instrument also obviates the regeneration step, allowing the binding kinetics between the green fluorescent protein and its antibody to be measured. In comparison to the conventional approach, the method simplifies the experimental procedure, reduces costs associated with sensor chips and biological samples, expedites kinetic measurements, and allows affinity constants to be determined more straightforwardly.

Label-free measurement of the yeast short chain TAG lipase activity by ESI-MS after one-step esterification

Triacylglycerol (TAG) lipases hydrolyze ester bonds in TAG and release diacylglycerol (DAG), monoacylglycerol (MAG), and FA. We present a one-step chemical derivatization method for label-free quantification of a mixture of TAG, DAG, and MAG following lipase assay by ESI-MS. Because the ionization efficiencies of TAG, DAG, and MAG are not identical, lipase reaction products, DAG and MAG, are derivatized to TAG species by esterifying their hydroxyl groups using acyl chloride, whose acyl chain contains one less (or one more) -CH2 group than that of substrate TAG. This resulted in three TAG species that were separated by 14 Da from one another and exhibited similar ion responses representing their molar amounts in the mass spectra. A good linear correlation was observed between peak intensity ratios and molar ratios in calibration curve. This method enables simultaneous quantification of TAG, DAG, and MAG in lipase assay and, in turn, allows stoichiometric determination of the concentrations of FAs released from TAG and DAG separately. By applying this strategy to measure both TAG and DAG lipolytic activities of the yeast Tgl2 lipase, we demonstrated its usefulness in studying enzymatic catalysis, as lipase enzymes often show dissimilar activities toward these lipids.

Improving performance of a rapid electrochemical MRSA assay: Optimisation of assay conditions to achieve enhanced discrimination of clinically important DNA sequences under ambient conditions

Electrochemical impedance spectroscopy (EIS) is a highly useful approach for the label free measurement of DNA hybridisation at functionalised electrode surfaces. Since label free detection relies upon a change in the electrochemical signal arising directly from the presence of target oligonucleotide or DNA/RNA sequences it is necessary to improve understanding of the conditions which produce a stable baseline value, promote optimal hybridisation of complementary sequences and can reduce non-specific binding effects. This study investigates both artificial DNA oligonucleotide sequences and clinical samples of MRSA genomic DNA, initially demonstrating that the use of tris(2-carboxyethyl)phosphine (TCEP) during probe layer formation improves both the initial baseline signal reproducibility and also the magnitude of the response upon hybridisation with a complementary target. Having demonstrated enhanced performance from TCEP modified electrodes, the assay is then used to detect clinical samples of MRSA. It is shown that improved performance is achieved both in terms of signal magnitude and discrimination against negative controls. Finally, formamide is added to the EIS measurement buffer and it is demonstrated that EIS measurement is possible in the presence of high formamide concentrations and that non-specific binding is also reduced under such conditions. The importance of these findings lies in the design of future electrochemical assays for nucleic acid biomarkers which are capable of functioning under ambient conditions but still have discriminatory power. A diagnostic device which does not have to operate at elevated temperatures will lead to increased simplicity and substantial battery and time savings which will further the widespread realisation of portable clinical diagnostic devices.

Defining Diagnostic Biomarkers Using Shotgun Proteomics and MALDI-TOF Mass Spectrometry.

Whole-cell MALDI-TOF has become a robust and widely used tool to quickly identify any pathogen. In addition to being routinely used in hospitals, it is also useful for low cost dereplication in large scale screening procedures of new environmental isolates for environmental biotechnology or taxonomical applications. Here, I describe how specific biomarkers can be defined using shotgun proteomics and whole-cell MALDI-TOF mass spectrometry. Based on MALDI-TOF spectra recorded on a given set of pathogens with internal calibrants, m/z values of interest are extracted. The proteins which contribute to these peaks are deduced from label-free shotgun proteomics measurements carried out on the same sample. Quantitative information based on the spectral count approach allows ranking the most probable candidates. Proteogenomic approaches help to define whether these proteins give the same m/z values along the whole taxon under consideration or result in heterogeneous lists. These specific biomarkers nicely complement conventional profiling approaches and may help to better define groups of organisms, for example at the subspecies level.

Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence measurements.

We report the results of a label-free analysis of ribonuclease activity using droplet-based microfluidics. The ribonucleolytic activity of ribonucleases (RNases) plays a critical role in cellular functions such as development, survival, growth and differentiation. Altered ribonucleolytic activity and/or the expression level of the RNase A family are known to be associated with pancreatic, bladder, ovarian and thyroid cancers among others. For this reason, the RNase A family is a meaningful protein biomarker that can be used in the diagnosis of cancer and as a target for new drug screening. There are some successful traditional methods for analysing the RNase activity, such as radioactive label-based assay, methylene blue-based assay, gel zymography, as well as other more recently developed methods such as electrochemical assay and fluorescence resonance energy transfer (FRET). However, these methods require analytical samples with a volume ranging from microliters to milliliters, and are not suitable for high-throughput analysis. Therefore, we integrated ethidium bromide (EtBr), which intercalates the chemical itself to nucleic acid, to droplet-based microfluidics for a cost-effective, high-throughput analysis. Put simply, this method is dependent on the amount of intercalated EtBr molecules on RNA. Our assay also uses visible light that is harmless to humans, unlike previous methods that used harmful UV rays, to excite the EtBr molecules. Specifically, we monitored the ribonucleolytic activity of less than 10 nM RNase A in droplets of about 330 picoliters. Also, half the maximal inhibitory concentration (IC50) of the RNase inhibitor was successfully measured in the same volume of droplets at a frequency of 40 hertz.

Integration of Quartz Crystal Microbalance-Dissipation and Reflection-Mode Localized Surface Plasmon Resonance Sensors for Biomacromolecular Interaction Analysis

The combination of label-free, surface-sensitive measurement techniques based on different physical principles enables detailed characterization of biomacromolecular interactions at solid-liquid interfaces. To date, most combined measurement systems have involved experimental techniques with similar probing volumes, whereas the potential of utilizing techniques with different surface sensitivities remains largely unexplored, especially for data interpretation. Herein, we report a combined measurement approach that integrates a conventional quartz crystal microbalance-dissipation (QCM-D) setup with a reflection-mode localized surface plasmon (LSPR) sensor. Using this platform, we investigate vesicle adsorption on a titanium oxide-coated sensing substrate along with the amphipathic, α-helical (AH) peptide-induced structural transformation of surface-adsorbed lipid vesicles into a supported lipid bilayer (SLB) as a model biomacromolecular interaction. While the QCM-D and LSPR signals both detected mass uptake arising from vesicle adsorption, tracking the AH peptide-induced structural transformation revealed more complex measurement responses based on the different surface sensitivities of the two techniques. In particular, the LSPR signal recorded an increase in optical mass near the sensor surface which indicated SLB formation, whereas the QCM-D signals detected a significant loss in net acoustic mass due to excess lipid and coupled solvent leaving the probing volume. Importantly, these measurement capabilities allowed us to temporally distinguish the process of SLB formation at the sensor surface from the overall structural transformation process. Looking forward, these label-free measurement capabilities to simultaneously probe adsorbates at multiple length scales will provide new insights into complex biomacromolecular interactions.

Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye.

Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level.