Self-folding deep cavitands embedded in a supported lipid bilayer are capable of recognizing suitably labeled proteins at the bilayer interface. The addition of a choline derived binding “handle” to a number of different proteins allows their selective noncovalent recognition, with association constants on the order of 105 M–1. The proteins are displayed at the water:bilayer interface, and a single binding handle allows recognition of the large, charged protein by a small molecule synthetic receptor via complementary shape and charge interactions.

Recent advances in graphene-based sensors have shown that heavily oxidized (GO) and reduced graphene oxide (rGO) are attractive materials for environmental sensing due to their unique chemical and physical properties. We describe here the fabrication of nanostructured GO assemblies with Ag nanoprisms for improved detection with surface enhanced Raman scattering (SERS). Specifically, 100-μm-sized, periodic-nanoprism-array domains were fabricated on top of the GO layers by GO-assisted lithography (GOAL). The atomically thin GO underlayers are shown to attract cyclic aromatic molecules to the surface, likely via π–π stacking interactions. The close proximity of the analyte to GO and nanoprism (NP) tips effectively suppresses fluorescent background and affords a plausible tertiary enhancement of photon emissions via an electron charge transfer (CT) process. The adsorption of analyte to rGO-NP leads to the appearance and/or shift of several Raman bands, which provided a means to gain molecular insights into the graphene-enhanced scattering process. The analytical merits were characterized with model compound Rhodamine 6G, where the detection limit could reach subnanomolar concentrations. The nanoprism GO substrates also prove effective for SERS multiplex measurement of several legacy aromatic pollutants. Three tetrachlorobiphenyl isomers could be identified from a mixture using their autonomous nonoverlapping molecular fingerprints, and the substrate offers remarkable trace detection of 2,2′,3,3′-tetrachlorobiphenyl (PCB-77).Keywords: graphene oxide; graphene-enhanced Raman spectroscopy (GERS); nanoprisms; polychlorinated biphenyls; surface-enhanced Raman spectroscopy (SERS);

Three-dimensional (3D) printing has undergone an exponential growth in popularity due to its revolutionary and near limitless manufacturing capabilities. Recent trends have seen this technology utilized across a variety of scientific disciplines, including the measurement sciences, but precise fabrication of optical components for high-performance biosensing has not yet been demonstrated. We report here 3D printing of high-quality, custom prisms by stereolithography that enable Kretschmann-configured plasmonic sensing of bacterial toxins. Simple benchtop polishing procedures render a smooth surface that supports propagation of surface plasmon polaritons with a deposited gold layer, which exhibit high bulk refractive index sensitivities and are capable of discriminating trace levels of cholera toxin on a supported lipid membrane interface. Further evidence of the flexibility of this manufacturing technique is demonstrated with printed prisms of varied geometries and in situ monitoring of nanoparticle growth by total internal reflection spectroscopy. This work represents the first example of 3D printed light-guiding sensing platforms and demonstrates the versatility and broad perspective of 3D printing in optical detection.

A novel bioconjugation strategy leading to ultrastable gold nanoparticles (AuNPs), utilizing DNA linkers and diluents in place of traditional self-assembled monolayers, is reported. The protective capacity of DNA confers straightforward biomolecular attachment and multistep derivatization capabilities to these nanoparticles and, more significantly, substantially enhances their stability in demanding and complex sensing environments. The DNA/AuNPs were assembled through pH-assisted thiol-gold bonding of single stranded DNA and salt aging, with preconjugated biotin moieties facing outward from the gold surface. These nanoparticles remain a stable colloidal suspension under a wide range of buffers and ionic strengths and can endure multiple rounds of lyophilization while retaining high biological activity. Furthermore, the high stability of the DNA/AuNPs allows for multiple reactions and conjugations to be performed within the colloidal suspensions (i.e., Protein A and antibody binding) for tailored and specific recognition to take place. We have demonstrated the applications of the DNA/AuNPs for colorimetric assays and ELISA feasibility; additionally, SPR imaging analysis of a supported membrane microarray shows excellent results with DNA/AuNPs as the enhancing agent. Together, the properties imparted by this interface render the material suitable for clinical and point-of-care applications where stability, throughput, and extended shelf lives are needed.

Electrospun nanofibers of a polyaniline (PANi)/(+)-camphor-10-sulfonic acid (HCSA)/poly(ethylene oxide) (PEO) composite doped with different variants of graphene oxide (GO) were fabricated and evaluated as chemiresistor gas sensors operating at room temperature. A new strategy for enhancing PANi/PEO gas sensor performance is demonstrated using GO dopants reduced via thermal (trGO) or chemical (crGO) routes. By varying the chemical reduction duration (6 h, crGO-6 or 24 h, crGO-24), tunable enhancement of sensor response was achieved. Upon exposure to short-chain aliphatic alcohol vapors, the partially reduced crGO-6 dopant exhibited higher response than GO and crGO-24, suggesting that the dopant enhances sensor performance via increased electrical conductivity over neat GO, and enhanced hydrogen bonding capability over the further-reduced crGO-24 variant. Sensor arrays consisting of PANi/PEO doped with trGO, crGO-6 or crGO-24 moieties successfully identified methanol, ethanol, and 1-propanol vapors using principal component analysis (PCA).

Quantification of β-1,4-galactosyltransferase (β-1,4-GT) activity is of considerable significance in the diagnosis of various cancers including lung and ovarian cancer. We report here the use of synthetic β-N-acetylglucosamine (NAG) ligands that contain hydrazide functional groups to determine galactosyltransferase activity by mass spectrometry. With hydrazide-linked β-D-NAG as the acceptor, the activity of β-1,4-GT is quantified by matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) with high efficiency. Using the disulfide moiety in a 3,3′-dithiodipropionic acid dihydrazide (DTP)-linked β-D-NAG probe, Au nanoparticles (AuNPs) are employed for enriching DTP-linked β-D-NAG after enzymatic reaction, and the ligand-bound AuNPs are subsequently deposited on a MALDI plate for analysis. In addition, we have demonstrated that a perfluorocarbon (PF) labeled β-NAG-ligand can be useful for surface-based enzymatic reaction with a perfluorooctadecanethiol (PFDT)-covered gold surface. Using the ratiometric method, the conversion rate of β-1,4-GT is determined to be 0.83 ± 0.03, which shows a high level of activity. This is the first work that uses hydrazide-linked β-D-NAG for activity analysis, providing a new surface-based MS approach to determine enzyme activity in a potentially high-throughput manner.

A hybrid material of gold nanodiscs on a calcinated titania nanofilm that allows for selective quantitative and qualitative characterization of surface-enriched phosphopeptides has been designed and reported. Fabrication was realized through a combination of layer-by-layer deposition and high temperature calcination for the titania, and hole-mask colloidal lithography for the plasmonic nanostructures. The morphology of the resulting titania material was rigorously characterized, exhibiting substantially decreased surface roughness, which allows for lithographic fabrication of plasmonic nanostructures. Moreover, high specificity in adsorption and enrichment of phosphopeptides was exhibited, which was verified by LSPR shifts and matching peaks under mass spectrometric analysis. The construction of these biochips should inform other combinatorial nanofabrication techniques, in addition to allowing future phosphoproteomic analyses to be performed in a time and resource-efficient manner.

Self-assembly of supramolecular structures has become an attractive means to create new biologically inspired materials and interfaces. We report the first robust hybrid bilayer systems readily coassembled from amphiphilic dendrimers and a naturally occurring phospholipid. Both concentration and generation of the dendrimers have direct impacts on the biophysical properties of the coassemblies. Raising the dendrimer concentration increases the hybrid bilayer stability, while changes in the generation and the concentration of the embedded dendrimers impact the fluidity of the coassembled systems. Multivalent dendrimer amine terminals allow for nondestructive in situ derivatization, providing a convenient approach to decorate and modulate the local environment of the hybrid bilayer. The coassembly of lipid/dendrimer interfaces offers a unique platform for the creation of hybrid systems with modular and precisely controllable behavior for further applications in sensing and drug delivery.Keywords: amphiphilic dendrimers; dendrimer/lipid hybrid membrane; self-assembly; supramolecular coassembly; surface plasmon resonance;

A patterned gold nanoparticle microarray, functionalized with a nanoscale silicate coating, has been developed for on-chip, high-throughput mass spectrometric analyses of biomolecules with minimal sample preparation and reagent costs. Fabrication was realized by the combination of layer-by-layer functionalization of the nanoparticles with suitable polyelectrolytes, followed by fluidic patterning of the glass microarray support and calcination for permanent fixation of the nano-coating. Performance of the microarray was evaluated for surface-assisted laser-desorption/ionization mass spectrometry (SALDI-MS), where the nano-silicate coating was found to enhance SALDI efficiency, resulting in comparable performance to some common organic matrices for small and medium sized molecules. Performance contributing factors of this material have been discussed; heat confinement and interband transition/plasmonic resonance may play important roles. Taking the accessibility of fabrication, performance, and reusability of this substrate together, the material developed here provides a new tool for multiplexed and chip-based mass spectrometric analysis.

Quantitation of alpha-glucosidase (α-GD) activity is of significance to diagnosis of many diseases including Pompe disease and type II diabetes. We report here a new method to determine α-GD activity using matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) in combination with carbohydrate microarray and affinity surface chemistry. Carbohydrate probes are synthesized for capture of the enzymatic reaction products and the adducts are loaded onto a fluorinated gold surface to generate an array, which is followed by characterization by MALDI-TOF-MS. The ratio of intensities is used to determine the level of activity of several enzymes. In addition, half maximal inhibitory concentration (IC50) of acarbose and epigallocatechin gallate are also determined using this approach, and the results agree well with the reported values. This method is advantageous as compared to conventional colorimetric techniques that typically suffer matrix interference problems from samples. The use of the polyfluorinated surface has effectively suppressed the interference.Keywords: alpha-glucosidase; carbohydrate; fluorinated surface; MALDI-MS; microarray

•MoS2/Ag nanohybrid was applied as a novel matrix in negative-ion MALDI-TOF MS.•The MoS2/Ag nanohybrid exerted synergistic effect on the detection of small molecules.•The MoS2/Ag nanohybrid showed good signal reproducibility and low background interferences comparing to organic matrices.•MoS2/Ag allows simultaneous analysis of multiple drugs and quantification of acetylsalicylic acid in spiked serum samples.This paper reports a facile synthesis of molybdenum disulfide nanosheets/silver nanoparticles (MoS2/Ag) hybrid and its use as an effective matrix in negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The nanohybrid exerts a strong synergistic effect, leading to high performance detection of small molecule analytes including amino acids, peptides, fatty acids and drugs. The enhancement of laser desorption/ionization (LDI) efficiency is largely attributed to the high surface roughness and large surface area for analyte adsorption, better dispersibility, increased thermal conductivity and enhanced UV energy absorption as compared to pure MoS2. Moreover, both Ag nanoparticles and the edge of the MoS2 layers function as deprotonation sites for proton capture, facilitating the charging process in negative ion mode and promoting formation of negative ions. As a result, the MoS2/Ag nanohybrid proves to be a highly attractive matrix in MALDI-TOF MS, with desired features such as high desorption/ionization efficiency, low fragmentation interference, high salt tolerance, and no sweet-spots for mass signal. These characteristic properties allowed for simultaneous analysis of eight different drugs and quantification of acetylsalicylic acid in the spiked human serum. This work demonstrates for the first time the fabrication and application of a novel MoS2/Ag hybrid, and provides a new platform for use in the rapid and high throughput analysis of small molecules by mass spectrometry.

Electrochemical biosensing represents a collection of techniques that may be utilized for capture and detection of biomolecules in both simple and complex media. While the instrumentation and technological aspects play important roles in detection capabilities, the interfacial design aspects are of equal importance, and often, those inspired by nature produce the best results. This review highlights recent material designs, recognition schemes, and method developments as they relate to targeted electrochemical analysis for biological systems. This includes the design of electrodes functionalized with peptides, proteins, nucleic acids, and lipid membranes, along with nanoparticle mediated signal amplification mechanisms. The topic of hyphenated surface plasmon resonance assays is also discussed, as this technique may be performed concurrently with complementary and/or confirmatory measurements. Together, smart materials and experimental designs will continue to pave the way for complete biomolecular analyses of complex and technically challenging systems.

The fabrication of large-scale, solid-supported lipid bilayer (SLB) arrays has traditionally been an arduous and complex task, primarily due to the need to maintain SLBs within an aqueous environment. In this work, we demonstrate the use of trehalose vitrified phospholipid vesicles that facilitate on-demand generation of microarrays, allowing each element a unique composition, for the label-free and high-throughput analysis of biomolecular interactions by SPR imaging (SPRi). Small, unilamellar vesicles (SUVs) are suspended in trehalose, deposited in a spatially defined manner, with the trehalose vitrifying on either hydrophilic or hydrophobic SPR substrates. SLBs are subsequently spontaneously formed on-demand simply by in situ hydration of the array in the SPR instrument flow cell. The resulting SLBs exhibit high lateral mobility, characteristic of fluidic cellular lipid membranes, and preserve the biological function of embedded cell membrane receptors, as indicated by SPR affinity measurements. Independent fluorescence and SPR imaging studies show that the individual SLBs stay localized at the area of deposition, without any encapsulating matrix, confining coral, or boundaries. The introduced methodology allows individually addressable SLB arrays to be analyzed with excellent label-free sensitivity in a real-time, high-throughput manner. Various protein–ganglioside interactions have been selected as a model system to illustrate discrimination of strong and weak binding responses in SPRi sensorgrams. This methodology has been applied toward generating hybrid bilayer membranes on hydrophobic SPR substrates, demonstrating its versatility toward a range of surfaces and membrane geometries. The stability of the fabricated arrays, over medium to long storage periods, was evaluated and found to be good. The highly efficient and easily scalable nature of the method has the potential to be applied to a variety of label-free sensing platforms requiring lipid membranes for high-throughput analysis of their properties and constituents.Keywords: devitrification; HBM; microarray; SLB; SPR; SPR imaging

Localized surface plasmon resonance (LSPR) represents a sensitive and versatile method for detection of biomolecules in a label-free fashion, but identification of bound analytes can be challenging with LSPR alone, especially for samples in a complex medium. We report the fabrication of an optically active, plasmonic film of gold nanoparticles by using a self-assembly and calcination process, which offers orthogonal measurements enabling multifaceted characterization on the same surface with LSPR and surface-assisted laser desorption/ionization mass spectrometry. This proof-of-concept study involves plasmonic characterization of the fabricated nanofilm, real-time monitoring of vesicle–surface interactions toward formation of fluid lipid bilayer, and mass spectrometric analysis of peptides and cytochrome c digest. This multifunction-enabling surface material can yield complementary analytical information, providing new tools for comprehensive analysis of biomolecular samples.

Optical sensors capable of colorimetric visualization and/or fluorescence detection have shown tremendous potential for field technicians and emergency responders, owing to the portability and low cost of such devices. Polydiacetylene (PDA)-enhanced nanofibers are particularly promising due to high surface area, facile functionalization, simple construction, and the versatility to empower either colorimetric or fluorescence signaling. We demonstrate here a dual-mode optical sensing with electrospun nanofibers embedded with various PDAs. The solvent-dependent fluorescent transition of nanofibers generated a pattern that successfully distinguished four common organic solvents. The colorimetric and fluorescent sensing of biotin–avidin interactions by embedding biotinylated-PCDA monomers into silica-reinforced nanofiber mats were realized for detection of biomolecules. Finally, a PDA-based nanofiber sensor array consisting of three monomers has been fabricated for the determination and identification of organic amine vapors using colorimetry and principal component analysis (PCA). The combination of PCA and the strategy of probing analytes in two different concentration ranges (ppm and ppth) led to successful analysis of all eight amines.

Self-folding deep cavitands embedded in a supported lipid bilayer are capable of recognizing suitably labeled proteins at the bilayer interface. The addition of a choline derived binding “handle” to a number of different proteins allows their selective noncovalent recognition, with association constants on the order of 105 M–1. The proteins are displayed at the water:bilayer interface, and a single binding handle allows recognition of the large, charged protein by a small molecule synthetic receptor via complementary shape and charge interactions.

We report here a surface plasmon resonance (SPR) method for detection of cell membrane binding proteins with high degree signal amplification carried out in an all-aqueous condition. Ultrahigh detection sensitivity was achieved for a membrane-based biosensing interface through the use of functional gold nanoparticles (AuNP) in combination with in situ atom transfer radical polymerization (ATRP) reaction. Fusion of phosphatidylcholine vesicles on a calcinated SPR gold chip established a supported bilayer membrane in which cell receptor monosialoganglioside GM1 was embedded for capture of bacterial cholera toxin (CT). The surface-bound CT was recognized with biotinylated anti-CT, which was linked to the biotin-AuNP through an avidin bridge. The biotin-AuNP surface was functionalized with ATRP initiator that triggers localized growth of poly(hydroxyl-ethyl methacrylate) (PHEMA) brush, contributing to marked SPR signal enhancement and quantitative measurement of CT at very low concentrations. The resulting polymer film has been characterized by optical and atomic force microscopy. A calibration curve for CT detection has been obtained displaying a response range from 6.3 × 10–16 to 6.3 × 10–8 M with a detection limit of 160 aM (equivalent to ∼9500 molecules in 100 μL sample solution). Sensitive detection of biomolecules in complex medium has been conducted with CT-spiked serum, and the detection limit can be effectively improved by 6 orders of magnitude compared to direct measurement in serum. The combined AuNP/ATRP method reported here opens new avenues for ultrasensitive detection of proteins on delicate sensor interfaces constructed by lipid membranes or cell membrane mimics.

This paper details the first use of a self-folding deep cavitand on a gold surface. A sulfide-footed deep, self-folding cavitand has been synthesized, and its attachment to a cleaned gold surface studied by electrochemical and SPR methods. Complete monolayer formation is possible if the cavitand folding is templated by noncovalent binding of choline or by addition of space-filling thiols to cover any gaps in the cavitand adsorption layer. The cavitand is capable of binding trimethylammonium-tagged guests from an aqueous medium and can be deposited in 2 × 2 microarrays on the surface for characterization by SPR imaging techniques. When biotin-labeled guests are used, the cavitand:guest construct can recognize and immobilize streptavidin proteins from aqueous solution, acting as an effective supramolecular biosensor for monitoring protein recognition.

We report the development of photocatalytically patterned TiO2 arrays for selective on-plate enrichment and direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of phosphopeptides. A thin TiO2 nanofilm with controlled porosity is prepared on gold-covered glass slides by a layer-by-layer (LbL) deposition/calcination process. The highly porous and rough nanostructure offers high surface area for selective binding of phosphorylated species. The patterned arrays are generated using an octadecyltrichlorosilane (OTS) coating in combination of UV irradiation with a photomask, followed by NaOH etching. The resulting hydrophilic TiO2 spots are thus surrounded by a hydrophobic OTS layer, which can facilitate the enrichment of low-abundance components by confining a large volume sample into a small area. The TiO2 arrays exhibit high specificity toward phosphopeptides in complex samples including phosphoprotein digests and human serum, and the detection can be made in the fmole range. Additional advantages of the arrays include excellent stability, reusability/reproducibility, and low cost. This method has been successfully applied to the analysis of phosphopeptides in nonfat milk. The patterned TiO2 arrays provide an attractive interface for performing on-plate reactions, including selective capture of target species for MALDI-MS analysis, and can serve as a versatile lab-on-a-chip platform for high throughput analysis in phosphoproteome research.

We report a novel optical platform based on SPR generation and confinement inside a defined three-dimensional microwell geometry that leads to background resonance-free SPR images. The array shows an exceptionally high signal-to-noise ratio (S/N > 80) for imaging analysis and subnanometric thickness resolution. An angular sensitivity of 1°/0.01 RIU has been achieved and the signal to background ratio (S/B) improves to 20, 1 order of magnitude higher than that of the best literature results. The design proves effective for probing-supported lipid membrane arrays in real time with a thickness resolution of 0.24 nm and allows for imaging analysis of microfluidic circuits where resonant spots are separated by only one pixel (∼7 μm). The high image quality and unique chip geometry open up new avenues for array screening and biomicrofluidics using SPRi detection.

We report the fabrication and characterization of gold-coated etched glass array substrates for surface plasmon resonance imaging (SPRi) analysis with significantly enhanced performance, in particular image contrast and sensitivity. The etching of the glass substrate induces a variation in the resonance condition and thus in the resonance angle between the etched wells and the surrounding area, leading to the isolation of the array spot resonance with a significant reduction of the background signal. FDTD simulations show arrays with large spots and minimal spot-to-spot spacing yield ideal differential resonance conditions, which are verified by experimental results. Simulations also indicate the etched well structure exhibits enhanced SPR electric field intensity by 3-fold as compared to standard planar gold chips. Changes in the bulk sensitivity of the etched arrays have been obtained at the 10–4 RIU level based on image intensity difference. The strong image contrast allows for improved microarray imaging analysis with easily distinguished signals from background resonance. The etched array chips are demonstrated for SPRi detection of bacterial toxins through the coating of an ultrathin SiO2 film for direct vesicle fusion that establishes a supported membrane-based biosensing interface. Protein detection with cholera toxin (CT) at 5 nM is obtained, making this chip one of the most sensitive SPR imaging substrates ever reported without a postbinding amplification scheme. Furthermore, the surface can be regenerated by Triton X-100 for repeated cycles of membrane formation, protein binding, and biomolecular removal. The reusability and enhanced performance of the etched glass array chips should find a broad range of applications, opening up new avenues for high-throughput SPR imaging detection with convenience and marked surface sensitivity.

Surface plasmon resonance (SPR)-based biosensing is one of the most advanced label free, real time detection technologies. Numerous research groups with divergent scientific backgrounds have investigated the application of SPR biosensors and studied the fundamental aspects of surface plasmon polaritons that led to new, related instrumentation. As a result, this field continues to be at the forefront of evolving sensing technology. This review emphasizes the new developments in the field of SPR-related instrumentation including optical platforms, chips design, nanoscale approach and new materials. The current tendencies in SPR-based biosensing are identified and the future direction of SPR biosensor technology is broadly discussed.

This paper reports the fabrication of solid-state nanofiber sensor arrays and their use for detection of multiple proteins using principal component analysis (PCA). Four cationic and anionic fluorescently embedded nanofibers are generated by an electrospinning method, yielding unique patterns of fluorescence change upon interaction with protein samples. Five metal and nonmetal containing proteins, i.e., hemoglobin, myoglobin, cytochrome c, BSA, and avidin, have been investigated; and the results show that distinct fluorescent patterns can be formed upon the addition of protein samples to the array of solid nanofiber substrates, allowing their unambiguous identification. The nanofiber films are highly repeatable with a batch-to-batch variation of approximately 5% and demonstrated outstanding reusability with less than a 15% loss of fluorescence intensity signal after 5 regenerations of test cycles. For a more practical visualization, a cluster map was generated using PCA of the change-in-fluorescence (ΔI) composite patterns, demonstrating the potential of the method for diagnostic applications.

Plasmon-waveguide resonance (PWR) sensors are particularly useful for the investigation of biomolecular interactions with or within lipid bilayer membranes. Many studies demonstrated their ability to provide unique qualitative information, but the evaluation of their sensitivity as compared to other surface plasmon resonance (SPR) sensors has not been broadly investigated. We report here a comprehensive sensitivity comparison of SPR and PWR biosensors for the p-polarized light component. The sensitivity of five different biosensor designs to changes in refractive index, thickness and mass are determined and discussed. Although numerical simulations show an increase of the electric field intensity by 30–35% and the penetration depth by four times in PWR, the waveguide-based method is 0.5–8-fold less sensitive than conventional SPR in all considered analytical parameters. The experimental results also suggest that the increase in the penetration depth in PWR is made at the expense of the surface sensitivity. The physical and structural reasons for PWR sensor limitations are discussed and a general viewpoint for designing more efficient SPR sensors based on dielectric slab waveguides is provided.

This paper details the incorporation of a water-soluble deep cavitand into a membrane bilayer assembled onto a nanoglassified surface for study of molecular recognition in a membrane-mimicking setting. The cavitand retains its host properties, and real-time analysis of the host:guest properties of the membrane:cavitand complex via surface plasmon resonance and fluorescence microscopy is described. The host shows selectivity for choline-derived substrates, and no competitive incorporation of substrate is observed in the membrane bilayer. A variety of trimethylammonium-derived substrates are suitable guests, displaying varied binding affinities in a millimolar range. The membrane:cavitand:guest complexes can be subsequently used to capture NeutrAvidin protein at the membrane surface if a biotin-derived guest molecule is used. The surface coverage of NeutrAvidin is affected by the spacer used to derivatize the biotin. Increased distance from the bilayer allows a higher concentration of protein to be immobilized, suggesting a diminishing detrimental steric effect when the binding event is shifted away from the surface.

We report a solid-state, nanofiber-based optical sensor for detecting proteins with an anionic fluorescent dendrimer (AFD). The AFD was encapsulated in cellulose acetate (CA) electrospun nanofibers, which were deacetylated to cellulose to generate secondary porous structures that are desirable for enhancing molecular interactions, and thus better signaling. The protein sensing properties of the fibers were characterized by monitoring the fluorescence response of cytochrome c (cyt c), hemoglobin (Hgb), and bovine serum albumin (BSA) as a function of concentration. Effective quenching was observed for the metalloproteins, cyt c and Hgb. The effect was primarily due to energy transfer of the imbedded fluorescent dendrimers to the protein, as both proteins contain heme portions. Electron transfer, caused through the electrostatic effects in the binding of the anionic dendrimer to the positive patches of globular proteins, could be responsible as well. BSA, on the other hand, triggered a “turn-on” response in fluorescence, suggesting the negatively charged BSA reduces the π−π stacking of the partially dispersed, negatively charged dendritic fluorophores through repulsion forces, which results in an increase in fluorescence. Stern−Volmer constants (Ksv) of the electrospun fibers were found to be 3.4 × 105 and 1.7 × 106 M−1 for cyt c and Hgb, respectively. The reusability of the nanofibers is excellent: the nanofibers demonstrated less than 15% change of fluorescence intensity signal in a 5-cycle test.Keywords: electrospinning; fluorescent dendrimers; nanofibers; protein biosensor

We report the use of silicate nanofilms for on-plate desalting and subsequently direct laser desorption/ionization-mass spectrometric (LDI-MS) analysis of peptides. A hydrophobic octadecyltrichlorosilane (OTS) monolayer is formed on a calcinated nanofilm on a gold substrate to facilitate sample deposition and interaction with the surface that allows effective removal of MS-incompatible contaminants such as salts and surfactants by simple on-plate washing while the peptides are retained on the spot. By elimination of interferences from matrix-related ions and contaminants, sensitivity of MS analysis has been enhanced over ca. 20 times, leading to improved detection of peptides at the low-femtomolar level. A high recovery rate of the peptides is obtained by using relatively rough nanofilms, which are prepared through a modified layer-by-layer deposition/calcination process. The performance of the films has been investigated with peptide samples in the presence of high salts (NaCl and sodium acetate) and urea. Compared to matrix-assisted laser desorption/ionization analysis with CHCA matrix, LDI with on-plate desalting offers marked improvement for analysis of peptides due to low background ions and reduction of sample complexity. Additionally, selective capture of the hydrophobic components of a protein can be achieved, providing a highly useful strategy for specific peptide enrichment. LDI with on-plate desalting approach has also been successfully applied to peptide analysis from protein digests.

We report a nanoscale calcinated silicate film fabricated on a gold substrate for highly effective, matrix-free laser desorption ionization mass spectrometry (LDI-MS) analysis of biomolecules. The calcinated film is prepared by a layer-by-layer (LbL) deposition/calcination process wherein the thickness of the silicate layer and its surface properties are precisely controlled. The film exhibits outstanding efficiency in LDI-MS with extremely low background noise in the low-mass region, allowing for effective analysis of low mass samples and detection of large biomolecules including amino acids, peptides, and proteins. Additional advantages for the calcinated film include ease of preparation and modification, high reproducibility, low cost, and excellent reusability. Experimental parameters that influence LDI on calcinated films have been systemically investigated. Presence of citric acid in the sample significantly enhances LDI performance by facilitating protonation of the analyte and reducing fragmentation. The wetting property and surface roughness appear to be important factors that manipulate LDI performance of the analytes. This new substrate presents a marked advance in the development of matrix-free mass spectrometric methods and is uniquely suited for analysis of biomolecules over a broad mass range with high sensitivity. It may open new avenues for developing novel technology platforms upon integration with existing methods in microfluidics and optics.

Ultrasensitive detection of proteins is of great importance to proteomics studies. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy by coupling a polymerization initiator to a biospecific interaction and inducing inline atom transfer radical polymerization (ATRP) for amplifying SPR response. Bacterial cholera toxin (CT) is chosen as the model protein that has been covalently immobilized on the surface for demonstrating the principle. The specific recognition is achieved by use of biotinylated anti-CT, which allows initiators with a biotin tag to be fixed at the protein binding site through a neutravidin bridge and triggers the localized growth of polymer brushes of poly(hydroxyl-ethyl methacrylate) (PHEMA) via an ATRP mechanism. To further enhance the signal, a second ATRP reaction is conducted that takes advantage of the hydroxyl groups of PHEMA brushes from the first step to form hyperbranched polymers onto the sensing surface. The two consecutive ATRP steps significantly improve SPR detection, allowing low amounts of CT that yield no direct measurement to be quantified with large signals. The resulting polymer film has been characterized by optical and atomic force microscopy. Ascorbic acid (AA) is employed as deoxygen reagent in the catalyst mixture that effectively suppresses oxygen interference, shortening the reaction time and making it possible for applying this ATRP approach to flow injection based SPR detection. A calibration curve of PHEMA amplification for CT detection based on surface coverage has been obtained that displays a correlation in a range from 8.23 × 10−15 to 3.61 × 10−12 mol/cm2 with a limit of detection of 6.27 × 10−15 mol/cm2. The versatile biotin−neutravidin interaction used here should allow adaptation of ATRP enhancement to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultrasensitive analysis of biomolecules with flow-injection assay and SPR spectroscopy.

Thin organic films with desirable redox properties have long been sought in biosensor research. We report here the development of a polymer thin film interface with well-defined hierarchical nanostructure and electrochemical behavior, and its characterization by electrochemical surface plasmon resonance (ESPR) spectroscopy. The nano-architecture build-up is monitored in real time with SPR, while the redox response is characterized by cyclic voltammetry in the same flow cell. The multilayer assembly is built on a self-assembled monolayer (SAM) of 1:1 (molar ratio) 11-ferrocenyl-1-undecanethiolate (FUT) and mercaptoundecanoic acid (MUA), and constructed using a layer-by-layer deposition of cationic poly(allylamine hydrochloride) (PAH) and anionic poly(sodium 4-styrenesulfonate) (PSS). Electron transfer (ET) on the mixed surface and the effect of the layer structures on ET are systematically studied. Under careful control, multiple layers can be deposited onto the 1:1 FUT/MUA SAM that presents unobstructed redox chemistry, indicating a highly ordered, extensively porous structure obtained under this condition. The use of SPR to trace the minute change during the electrochemical process offers neat characterization of local environment at the interface, in particular double layer region, allowing for better control over the redox functionality of the multilayers. The 1:1 SAM has a surface coverage of 4.1 ± 0.3 × 10−10 mol cm−2 for ferrocene molecules and demonstrates unperturbed electrochemistry activity even in the presence of a 13 nm polymer film adhered to the electrode surface. This thin layer possesses some desirable properties similar to those on a SAM while presenting ∼15 nm exceedingly porous structure for high loading capacity. The high porosity allows perchlorate to freely partition into the film, leading to high current density that is useful for sensitive electrochemical measurements.

Ever since the advent of surface plasmon resonance (SPR) and SPR imaging (SPRi) in the early 1990s, their use in biomolecular interaction analysis (BIA) has expanded phenomenally. An important research area in SPR sensor development is the design of novel and effective interfaces that allow for the probing of a variety of chemical and biological interactions in a highly selective and sensitive manner. A well-designed and robust interface is a necessity to obtain both accurate and pertinent biological information. This review covers the recent research efforts in this area with a specific focus towards biointerfaces, new materials for SPR biosensing, and novel array designs for SPR imaging. Perspectives on the challenges ahead and next steps for SPR technology are discussed.

The development of effective Escherichia coli (E. coli) sensors has been of great importance and urgent need to public health and safety in the wake of the spinach outbreak in California in 2006. We report here an enzymatic signal enhancement method for highly sensitive and fast detection of E. coli based on the generation of a mass-enhancing product at the sensing interface that is quantified by surface plasmon resonance (SPR) spectroscopy. The insoluble product is formed through the reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) with a horse radish peroxidase (HRP) conjugated antibody that attaches to the bacteria captured on a self-assembled monolayer (SAM) surface. Results indicate a signal enhancement of E. coli cells on the order of 250% as compared to the control samples. In addition, this signal enhancement was consistent for over six orders of concentration range. The limit of detection for E. coli is determined to be 103 cfu/mL by this method, offering excellent detection sensitivity for probing actual E. coli levels in produce and other real world samples. The method was further demonstrated for E. coli measurement in the complex matrix of spinach leaves where a dynamic range over three orders of magnitude of concentrations was obtained with a limit of detection of 104 cfu/mL. This approach is simple, fast and sensitive; given the relative simple procedure of adding an HRP tag to an antibody, it opens new route for detection of low levels of biomolecules, in particular bacteria and viruses, in a complex matrix.

Monitoring multiple biological interactions in a multiplexed array format has numerous advantages. However, converting well-developed surface chemistry for spectroscopic measurements to array-based high-throughput screening is not a trivial process and often proves to be the bottleneck in method development. This paper reports the fabrication and characterization of a new carbohydrate microarray with synthetic sialosides for surface plasmon resonance imaging (SPRi) analysis of lectin-carbohydrate interactions. Contact printing of functional sialosides on neutravidin-coated surfaces was carried out and the properties of the resulting elements were characterized by fluorescence microscopy and atomic force microscopy (AFM). Sambucus nigra agglutinin (SNA) was deposited on four different carbohydrate functionalized surfaces and differential binding was analyzed to reveal affinity variation as a function of headgroup sialic acid structures and linking bonds. SPRi studies indicated that this immobilization method could result in high quality arrays with RSD < 5% from array element to array element, superior to the conventional covalent linkage used for protein cholera toxin (CT) in a comparison experiment, which yields nonuniform array elements with RSD > 15%. Multiplexed detection of SNA/biotinylated sialoside interactions on arrays up to 400 elements has been performed with good data correlation, demonstrating the effectiveness of the biotin−neutravidin-based biointerface to control probe orientation for reproducible and efficient protein binding to take place. Additionally, the regeneration of the array surface was demonstrated with a glycine stripping buffer, rendering this interface reusable. This in-depth study of array surface chemistry offers useful insight into experimental conditions that can be optimized for better performance, allowing many different protein-based biointeractions to be monitored in a similar manner.Keywords: biointerface; biosensor; biotinylated sialosides; carbohydrate microarray; surface plasmon resonance imaging

We report a microfabrication approach to generate well-defined, addressable, and regenerable lipid membrane arrays in poly(dimethylsiloxane) (PDMS) microchips for label-free analysis of lipid−protein interactions with surface plasmon resonance imaging (SPRi). The multiplexed detection is demonstrated with a tethered bilayer membrane array built in parallel microchannels. These channels allow multiple measurements to be carried out simultaneously, showing low deviations for element-to-element variation in quantifiable signal. Lipid-conjugated receptors were utilized as model systems for protein binding analysis, and the feasibility of regenerating the tethering sublayer after binding was investigated. The results show that the lipid membrane can be removed effectively by nonionic surfactant Triton X-100. The small variance in SPR signal for the buildup process, i.e., <4% RSD for 3 cycles of detection, removal, and regeneration, indicates the sensing interface is highly reproducible. A calibration curve was obtained for cholera toxin using the monosialoganglioside (GM1) receptor, displaying a linear relationship in the 25 to 175 μg/mL range with a limit of detection of 260 nM. In addition, interaction of a phosphatidylinositol (PIP) with its binding protein and biotin/avidin interactions were employed for array measurements. To further enhance the SPR detection signal, a layer-by-layer amplification strategy was demonstrated that uses biotinylated antibody, NeutrAvidin and biotinylated anti-avidin, and the signal for protein binding on the membrane increased by 400%. The tethered membrane array technology, in combination with SPRi, offers an attractive platform for studies of membrane proteins, and can also find a range of applications for rapid screening of drug candidates interacting with proteins embedded in the near-native environment.

New sensing materials that are robust, biocompatible, and amenable to array fabrication are vital to the development of novel bioassays. Herein we report the fabrication of ultrathin (ca. 5−8 nm) glass (silicate) layers on top of a gold surface for surface plasmon resonance (SPR) biosensing applications. The nanoglass layers are fabricated by layer-by-layer (LbL) deposition of poly(allylamine) hydrochloride (PAH) and sodium silicate (SiOx), followed by calcination at high temperature. To deposit these layers in a uniform and reproducible manner, we employed a high-volume, low-pressure (HVLP) paint gun technique that offers high precision and better control through pressurized nitrogen gas. The new substrates are stable in solution for a long period of time, and scanning electron microscopy (SEM) images confirm that these films are nearly fracture-free. In addition, atomic force microscopy (AFM) indicates that the surface roughness of the silicate layers is low (rms = 2 to 3 nm), similar to that of bare glass slides. By tuning the experimental parameters such as HVLP gun pressure and layers deposited, different surface morphology could be obtained as revealed by fluorescence microscopy and SEM images. To demonstrate the utility of these ultrathin, fracture-free substrates, lipid bilayer membranes composed of phosphorylated derivatives of phosphoinositides (PIs) were deposited on the new substrates for biosensing applications. Fluorescence recovery after photobleaching (FRAP) data indicated that these lipid components in the membranes were highly mobile. Furthermore, interactions of PtdIns(4,5)P2 and PtdIns(4)P lipids with their respective binding proteins were detected with high sensitivity by using SPR spectroscopy. This method of glass deposition can be combined with already well-developed surface chemistry for a range of planar glass assay applications, and the process is amenable to automation for mass production of nanometer thick silicate chips in a highly reproducible manner for label-free measurements.

Gold nanoparticles (AuNPs) have been studied as a potential solid-state matrix for laser desorption/ionization mass spectrometry (LDI-MS) but the efficiency in ionization remains low. In this report, AuNPs are capped by a self-assembled monolayer of cysteamine and modified with α-cyano-4-hydroxycinnanic acid (CHCA) for effective MALDI measurements. CHCA-terminated AuNPs offer marked improvement on peptide ionization compared with citrate-capped or cysteamine-capped AuNPs. The coating also effectively suppresses formation of Au cluster ions and analyte fragment ions, leading to cleaner mass spectra. Addition of glycerol and citric acid to the peptide/AuNPs sample further improves the performance of these AuNPs for LDI-MS analysis. Glycerol appears to enhance the dispersion of AuNPs in sample spots, increasing the sample ionization and shot-to-shot reproducibility, while citric acid serves as an external proton donor, providing high production of protonated analyte ions and reducing fragmentation of peptides on the nanoparticle-based surface. Optimal ratios of citric acid, glycerol, and AuNPs in sample solution have been systematically studied. A more than 10-fold increase for desorption ionization of peptides can be achieved by combining 5% glycerol and 20 mM citric acid with the CHCA-terminated AuNPs. The applicability of the CHCA-AuNPs for LDI-MS analysis of protein digests has also been demonstrated. This work shows the potential of AuNPs for SALDI-MS analysis, and the improvement with chemical functionalization, controlled dispersion, and use of an effective proton donor.

Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein−carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin−carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin−NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that α2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 ± 93 nM for SNA binding to Neu5Acα2,6-LHEB. Transient SPR kinetics confirms the KD value from the equilibrium binding studies. A linear relationship was obtained in the 10−100 µg/mL range with limit of detection of ∼50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin−NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate−protein interactions in real time.

We report the development of an air-stable, supported membrane array by use of photolithography for label-free detection of lipid−protein interactions. Phosphoinositides and their phosphorylated derivatives (PIPs) were studied for their binding properties to proteins with lipid microarray in combination with surface plasmon resonance imaging (SPRi). We have demonstrated a simple method to fabricate lipid arrays using photoresist and carried out a series of surface characterizations with SPRi, ac impedance, cyclic voltammetry, and fluorescence microscopy to validate the array quality and lipid bilayer formation. A number of lipid compositions have been tested for the robustness of resulting membranes when undergoing dehydration and rehydration procedures, and the 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine/poly(ethylene glycol)-phosphatidylethanolamine (DOPC+/PEG-PE) system stands out as the best performer that yields the recovery to within 2% of the original state according to SPR sensorgrams. Limits of detection on the dehydrated/rehydrated DOPC+/PEG-PE membranes were determined to be 33 nM for avidin binding to biotinylated lipids, 73.5 nM for cholera toxin to GM1, and 25 nM for PtdIns(4,5)P2-binding protein (P4,5BP) to PtdIns(4,5)P2 lipid, respectively. These results demonstrate the suitability and sensitivity of this membrane for constructing membrane arrays for SPRi analysis under ambient conditions. With the use of this addressable and functional lipid membrane array, the screening of specific lipid−protein interactions has been conducted. Strong and specific interactions between P4,5BP and PtdIns(4,5)P2/DOPC+/PEG-PE membrane were observed as expected, while cross reactions were spotted for P4,5BP/PtdIns(4)P and avidin/GM1 at varied degrees. The air-stable membrane array demonstrated here presents a simple, effective approach for constructing functional membrane surfaces for screening applications, which opens a new avenue for the label-free study of membrane proteins and other forms of lipid−membrane interactions.

The interaction of pore-forming streptolysin O (SLO) with biomimetic lipid membranes has been studied by electrochemical methods. Phosphatidylcholine lipid vesicles were deposited onto gold electrodes modified with supporting layers of hexyl thioctate (HT) or thioctic acid tri(ethylene glycol) ester (TA-TEGE), and integrity and permeability of the resulting membranes were characterized by cyclic voltammetry and impedance spectroscopy. Both positively and negatively charged electrochemical probes, potassium ferrocyanide, hexaammineruthenium(III) chloride, and ferrocene carboxylic acid (FCA), were employed to evaluate their suitability to probe the membrane permeability properties, with FCA exhibiting ideal behavior and thus employed throughout the work. Fusion of vesicles incubated with SLO on the electrodes yielded membranes that showed a distinctive response pattern for FCA as a function of SLO concentration. A direct dependence of both the currents and peak separation of FCA in the cyclic voltammograms was observed over a concentration range of 0–10 hemolytic units (HU)/µL of the toxin. The interaction of SLO with preformed supported lipid membranes was also investigated, and much lower response was observed, suggesting a different extent of membrane−toxin interactions on such an interface. Nonionic surfactant Triton was found to disrupt the vesicle structure but could not completely remove a preformed membrane to fully restore the electrode response. The information reported here offers some unique insight into toxin−surface interactions on a hybrid membrane, facilitating the development of electrochemically based sensing platforms for detecting trace amounts of bacterial toxins via the perforation process.

Supported bilayer membranes (SBMs) formed on solid substrates, in particular glass, provide an ideal cell mimicking model system that has been found to be highly useful for biosensing applications. Although the stability of the membrane structures is known to determine the applicability, the subject has not been extensively investigated, largely because of the lack of convenient methods to monitor changes of membrane properties on glass in real time. This work reports the evaluation of the stability properties of a series of SBMs against chemical and air damage by use of surface plasmon resonance spectroscopy and nanoglassified gold substrates. Seven SBMs composed of phosphatidylcholine and DOPC+, including single-component, mixed, protein-reinforced SBMs (rSBMs) and protein-tethered bilayer membranes (ptBLMs), are studied. The stability properties under various conditions, especially the effects of surfactants, organic solvents, and dehydration damage on the bilayers, are compared. PC membranes are found to be easily removed from the glassy surfaces using relatively low concentrations of the surfactants, while DOPC+ is markedly more stable toward nonionic surfactant. DOPC+ membranes also demonstrated remarkable air stability while PC films exhibited considerable damage from dehydration. Doping of cholesterol does not improve PC’s stability against SDS and Triton but changes the lipid membrane packing enough to protect against dehydration damage. Although rSBMs and ptBLMs improve air stability to a certain degree, they are still quite susceptible to significant damage/removal from ionic and nonionic surfactants at lower concentrations. Overall, DOPC+ has noted higher stability on glass, likely due to the favorable electrostatic interaction between the silicate surface and the lipid headgroup, making it a good candidate for application. Nanoglassy SPR proves to be an attractive platform capable of rapidly screening film stability in real-time, providing critical information for future work using supported membranes for sensing applications.

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules.

We report the microfluidic fabrication of robust and fluid tethered bilayer arrays within a poly(dimethylsiloxane) (PDMS) chip, and demonstrate its addressability and biosensing by incorporating the GM1 receptor into the bilayer framework for detection of cholera toxin. Rapid optimization of the experimental conditions is achieved by using nanoglassified surfaces in combination with surface plasmon resonance. The ultrathin glassy film on gold mimics glass surfaces employed in microfluidics, allowing real-time monitoring of multiple assembly steps and therefore permitting rapid prototyping of microfluidic arrays. The tethered bilayer array utilizes a covalently immobilized biotinylated protein for generation of well-defined capture zones where a streptavidin link is employed for the immobilization of biotinylated vesicles. Fusion of captured vesicles is accomplished using a concentrated PEG solution, and the lateral diffusion of the tethered bilayer membrane is characterized by fluorescence recovery after photobleaching methods. The tethered membrane arrays demonstrate marked stability and high mobility, which provide an ideal host environment for membrane-associated proteins and open new avenues for high-throughput analysis of these proteins.

A novel amperometric sensor that integrates two independent measurement schemes into a single chip for detection of glucose is fabricated. The sensor uses micro-patterned Prussian blue (PB) and ferrocene modified glucose oxidase covered by a thin Nafion membrane. We have developed an amperometric sensor for the detection of glucose that integrates two measurement schemes into a single chip. For fabrication of the sensing interface, micro-contact printing was used to transfer a self-assembled monolayer template onto a gold substrate, allowing selective electrochemical deposition of a PB array. The protective layer of the PB array was subsequently removed and replaced with a layer of redox-functionalized glucose oxidase (GOx), while the entire surface was finally covered with a perm-selective GOx–Nafion membrane. A variety of surface analytical techniques, including atomic force microscopy, surface plasmon resonance imaging and spectroscopic ellipsometry were employed to characterize the composite PB array electrode. The hybrid sensing interface allowed amperometric measurements of glucose to be carried out with two independent schemes at different potentials. The cathodic current was obtained with the PB array functioning as the electrocatalyst, while the anodic current was measured at a higher potential via a mediation mechanism using the ferrocene-modified GOx. For the quantitative detection of glucose, flow-injection analysis was used, and both the operating conditions and the design parameters were optimized. Linear responses were obtained for both anodic and cathodic signals over a concentration range from 0.1 to 50 mM, with a detection limit of 75 µM. The specificity of the sensor was demonstrated with respect to ascorbic and lactic acid. The implementation of integrated detection mechanisms allows the independent measurement of amperometric signals at two separate potentials. This improves the information gathering and opens up new avenues for developing novel methods that potentially eliminate false signal readings.

We report on the use of PDMS multichannels for affinity studies of DNA aptamer–human Immunoglobulin E (IgE) interactions by surface plasmon resonance imaging (SPRi). The sensing surface was prepared with thiol-terminated aptamers through a self-assembling process in the PDMS channels defined on a gold substrate. Cysteamine was codeposited with the thiol aptamers to promote proper spatial arrangement of the aptamers and thus maintain their optimal binding efficiencies. Four aptamers with different nucleic acid sequences were studied to test their interaction affinity toward IgE, and the results confirmed that aptamer I (5′-SH-GGG GCA CGT TTA TCC GTC CCT CCT AGT GGC GTG CCC C-3′) has the strongest binding affinity. Control experiments were conducted with a PEG-functionalized surface and IgG was used to replace IgE in order to verify the selective binding of aptamer I to the IgE molecules. A linear concentration-dependent relationship between IgE and aptamer I was obtained, and a 2-nM detection limit was achieved. SPRi data were further analyzed by global fitting, and the dissociation constant of aptamer I–IgE complex was found to be 2.7 × 10−7 M, which agrees relatively well with the values reported in the literature. Aptamer affinity screening by SPR imaging demonstrates marked advantages over competing methods because it does not require labeling, can be used in real-time, and is potentially high-throughput. The ability to provide both qualitative and quantitative results on a multichannel chip further establishes SPRi as a powerful tool for the study of biological interactions in a multiplexed format.

A versatile and novel method has been developed for microfluidic immunosensing of the food-borne pathogen Staphylococcus enterotoxin B (SEB) in poly(dimethylsiloxane) (PDMS) chips. Supported bilayer membranes (SBMs) were generated by vesicle fusion in oxidized PDMS microchannels for minimizing non-specific adsorption of biomolecules. The stability of SBMs was strengthened with a streptavidin layer to make them air-stable and allow for subsequent display of the biotin-functionalized antibodies. The reinforced supported bilayer membranes (r-SBMs) are fluid, exhibiting a lateral diffusion coefficient of ∼1.9 µm2 s−1, and no detectable change of mobility was found after dehydration/rehydration. This is a substantial improvement over phosphatidylcholine (PC) membranes on PDMS, which suffered a roughly 10% reduction in the mobile fraction and 30% decrease in mobility after dehydration. Non-specific protein adsorption in the membrane-treated channels was reduced 100–1000 fold as compared to PDMS surfaces without a membrane coating. A flow-based microfluidic immunosensor for SEB was developed using antibodies linked to the r-SBMs in PDMS channels, and a detection limit of 0.5 ng mL−1 was obtained from the linear portion of the calibration curve. The microchip was applied to detection of SEB in milk, and similar response and sensitivity were obtained, demonstrating the sensor's remarkable performance for real world samples. The r-SBMs overcome the stability hurdle in SBM-modified surfaces, opening up possibilities for transport and storage of membrane-functionalized microchips in the dehydrated form without compromising the performance, and facilitating the commercialization of disposable SBM-based microdevices.

We report a nanoscale lipid membrane-based sensor of conjugated polydiacetylene (PDA) vesicles for fluorescence detection of organic amines. The vesicle sensor was constructed by incorporation of a BODIPY fluorescent dye into the PDA vesicles. The fluorescent properties of the resulting vesicles can be manipulated by adjusting lipid components, and are controlled by environmental and solution conditions. The fluorescence of the BODIPY dye was significantly quenched in the polymerization of diacetylene lipid vesicles by a UV irradiation process. However, it was sufficiently recovered by external stimuli such as a hike of solution pH. The fluorescence recovery process was reversible, and a decrease in solution pH resulted in repeated quenching. The reported system transforms an external stimulus into a large fluorescence intensity change, demonstrating great potential in developing new signal reporting method for biosensor design. The quench-recovery phenomenon of the BODIPY–PDA is believed to be related to the energy transfer between the dye and the PDA conjugate backbone. The vesicle sensor was applied for detecting an organic amine, triethylamine (TEA) and a large linear relationship was obtained between the increase in fluorescence intensity and the concentrations of TEA. The detection limit of TEA by vesicle sensors using fluorescence recovery was found to be 10 μM.

Morphological transformations of bolaamphiphilic polydiacetylene (L-Glu-Bis-3) lipid assemblies from helical ribbons to vesicles and flat sheets through controlled doping are described, and the role of specific lipid dopants in these processes is discussed. Upon doping with cell surface receptor GM1 ganglioside, fluid vesicular structures start to emerge, coexisting with the micro-crystalline helical ribbons. The vesicle formation is further facilitated and stabilized by the introduction of cholesterol into the system, presumably through surface curvature variation induced by inhomogeneous distribution and dynamic clustering of GM1 and cholesterol within the doped assemblies. Extended helical ribbons are ‘truncated’ into patches of flat sheets when a sufficient amount of Bis-1, a structurally compatible symmetric bolaamphiphilic diacetylene lipid, is doped. The results reaffirm the important roles of packing geometry and headgroup chirality in the formation of extended helical ribbon structures. The doped assemblies of bolaamphiphiles allow for capture of intermediate structures of morphological transformation using transmission electron microscopy (TEM). A vesicle-to-ribbon transformation mechanism via lateral reorganization within relatively fluid vesicular microstructures has been suggested. Understanding of the doping-induced transformation process provides useful information for the design of advanced materials where the microscopic morphology of material is crucial to its function.

Gold nanoparticles (AuNPs) have been studied as a potential solid-state matrix for laser desorption/ionization mass spectrometry (LDI-MS) but the efficiency in ionization remains low. In this report, AuNPs are capped by a self-assembled monolayer of cysteamine and modified with α-cyano-4-hydroxycinnanic acid (CHCA) for effective MALDI measurements. CHCA-terminated AuNPs offer marked improvement on peptide ionization compared with citrate-capped or cysteamine-capped AuNPs. The coating also effectively suppresses formation of Au cluster ions and analyte fragment ions, leading to cleaner mass spectra. Addition of glycerol and citric acid to the peptide/AuNPs sample further improves the performance of these AuNPs for LDI-MS analysis. Glycerol appears to enhance the dispersion of AuNPs in sample spots, increasing the sample ionization and shot-to-shot reproducibility, while citric acid serves as an external proton donor, providing high production of protonated analyte ions and reducing fragmentation of peptides on the nanoparticle-based surface. Optimal ratios of citric acid, glycerol, and AuNPs in sample solution have been systematically studied. A more than 10-fold increase for desorption ionization of peptides can be achieved by combining 5% glycerol and 20 mM citric acid with the CHCA-terminated AuNPs. The applicability of the CHCA-AuNPs for LDI-MS analysis of protein digests has also been demonstrated. This work shows the potential of AuNPs for SALDI-MS analysis, and the improvement with chemical functionalization, controlled dispersion, and use of an effective proton donor.AuNPs capped by a self-assembled monolayer of cysteamine and modified with CHCA for effective LDI.Download high-res image (125KB)Download full-size image