Aaron R. Halpern

Single-Nanoparticle Near-Infrared Surface Plasmon Resonance Microscopy for Real-Time Measurements of DNA Hybridization Adsorption

A novel 814 nm near-infrared surface plasmon resonance (SPR) microscope is used for the real-time detection of the sequence-selective hybridization adsorption of single DNA-functionalized gold nanoparticles. The objective-coupled, high numerical aperture SPR microscope is capable of imaging in situ the adsorption of single polystyrene and gold particles with diameters ranging from 450 to 20 nm onto a 90 μm × 70 μm area of a gold thin film with a time resolution of approximately 1-3 s. Initial real-time SPR imaging (SPRI) measurements were performed to detect the accumulation of 40 nm gold nanoparticles for 10 min onto a gold thin film functionalized with a 100% complementary DNA surface at concentrations from 5 pM to 100 fM by counting individual particle binding events. A 100% noncomplementary DNA surface exhibited virtually no nanoparticle adsorption. In contrast, in a second set of SPRI measurements, two component complementary/noncomplementary mixed DNA monolayers that contained a very small percentage of complementary sequences ranging from 0.1 to 0.001%, showed both permanent and transient hybridization adsorption of the gold nanoparticles that could be tracked both temporally and spatially with the SPR microscope. These experiments demonstrate that SPR imaging measurements of single biofunctionalized nanoparticles can be incorporated into bioaffinity biosensing methods at subpicomolar concentrations.

Lithographically Patterned Electrodeposition of Gold, Silver, and Nickel Nanoring Arrays with Widely Tunable Near-Infrared Plasmonic Resonances

A novel low-cost nanoring array fabrication method that combines the process of lithographically patterned nanoscale electrodeposition (LPNE) with colloidal lithography is described. Nanoring array fabrication was accomplished in three steps: (i) a thin (70 nm) sacrificial nickel or silver film was first vapor-deposited onto a plasma-etched packed colloidal monolayer; (ii) the polymer colloids were removed from the surface, a thin film of positive photoresist was applied, and a backside exposure of the photoresist was used to create a nanohole electrode array; (iii) this array of nanoscale cylindrical electrodes was then used for the electrodeposition of gold, silver, or nickel nanorings. Removal of the photoresist and sacrificial metal film yielded a nanoring array in which all of the nanoring dimensions were set independently: the inter-ring spacing was fixed by the colloidal radius, the radius of the nanorings was controlled by the plasma etching process, and the width of the nanorings was controlled by the electrodeposition process. A combination of scanning electron microscopy (SEM) measurements and Fourier transform near-infrared (FT-NIR) absorption spectroscopy were used to characterize the nanoring arrays. Nanoring arrays with radii from 200 to 400 nm exhibited a single strong NIR plasmonic resonance with an absorption maximum wavelength that varied linearly from 1.25 to 3.33 μm as predicted by a simple standing wave model linear antenna theory. This simple yet versatile nanoring array fabrication method was also used to electrodeposit concentric double gold nanoring arrays that exhibited multiple NIR plasmonic resonances.

Surface plasmon resonance phase imaging measurements of patterned monolayers and DNA adsorption onto microarrays.

The optical technique of surface plasmon resonance phase imaging (SPR-PI) is implemented in a linear microarray format for real-time measurements of surface bioaffinity adsorption processes. SPR-PI measures the phase shift of p-polarized light incident at the SPR angle reflected from a gold thin film in an ATR Kretschmann geometry by creating an interference fringe image on the interface with a polarizer-quartz wedge depolarizer combination. The position of the fringe pattern in this image changes upon the adsorption of biomolecules to the gold thin film. By using a linear array of 500 μm biosensor element lines that are perpendicular to the interference fringe image, multiple bioaffinity adsorption measurements can be performed in real time. Two experiments were performed to characterize the sensitivity of the SPR-PI measurement technique: First, a ten line pattern of a self-assembled monolayer of 11-mercaptoundecamine (MUAM) was created via photopatterning to verify that multiple phase shifts could be measured simultaneously. A phase shift difference (Δφ) of Δφ = 182.08 ± 0.03° was observed for the 1.8 nm MUAM monolayer; this value agrees with the phase shift difference calculated from a combination of Fresnel equations and Jones matrices for the depolarizer. In a second demonstration experiment, the feasibility of SPR-PI for in situ bioaffinity adsorption measurements was confirmed by detecting the hybridization and adsorption of single stranded DNA (ssDNA) onto a six-component DNA line microarray patterned monolayer. Adsorption of a full DNA monolayer produced a phase shift difference of Δφ = 28.80 ± 0.03° at the SPR angle of incidence and the adsorption of the ssDNA was monitored in real time with the SPR-PI. These initial results suggest that SPR-PI should have a detection limit roughly 100 times lower than traditional intensity-based SPR imaging measurements.

Near Infrared Surface Plasmon Resonance Phase Imaging and Nanoparticle-Enhanced Surface Plasmon Resonance Phase Imaging for Ultrasensitive Protein and DNA Biosensing with Oligonucleotide and Aptamer Microarrays

The techniques of surface plasmon resonance-phase imaging (SPR-PI) and nanoparticle-enhanced SPR-PI have been implemented for the multiplexed bioaffinity detection of proteins and nucleic acids. The SPR-PI experiments utilized a near-infrared 860 nm light emitting diode (LED) light source and a wedge depolarizer to create a phase grating on a four-element single-stranded DNA (ssDNA) microarray; bioaffinity adsorption onto the various microarray elements was detected via multiplexed real time phase shift measurements. In a first set of demonstration experiments, an ssDNA aptamer microarray was used to directly detect thrombin at concentrations down to 100 pM with SPR-PI. Two different ssDNA aptamers were used in these experiments with two different Langmuir adsorption coefficients, K(A1) = 4.4 × 10(8) M(-1) and K(A2) = 1.2 × 10(8) M(-1). At concentrations below 1 nM, the equilibrium phase shifts observed upon thrombin adsorption vary linearly with concentration with a slope that is proportional to the appropriate Langmuir adsorption coefficient. The observed detection limit of 100 pM is approximately 20 times more sensitive than that observed previously with SPRI. In a second set of experiments, two short ssDNA oligonucleotides (38mers) were simultaneously detected at concentrations down to 25 fM using a three-sequence hybridization format that employed 120 nm DNA-modified silica nanoparticles to enhance the SPR-PI signal. In this first demonstration of nanoparticle-enhanced SPR-PI, the adsorbed silica nanoparticles provided a greatly enhanced phase shift upon bioaffinity adsorption due to a large increase in the real component of the interfacial refractive index from the adsorbed nanoparticle. As in the case of SPR-PI, the detection limit of 25 fM for nanoparticle-enhanced SPR-PI is approximately 20 times more sensitive than that observed previously with nanoparticle-enhanced SPRI.

On-Chip Synthesis of Protein Microarrays from DNA Microarrays Via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of mRNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and antiluciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications.

Electrodeposition of Polydopamine Thin Films for DNA Patterning and Microarrays

The controlled electrodeposition of functional polydopamine (PDA) thin films from aqueous dopamine solutions is demonstrated with a combination of electrochemistry, atomic force microscopy (AFM), and surface plasmon resonance (SPR) measurements. PDA micropatterns are then fabricated by electrodeposition on micrometer length scale gold electrodes and used for attaching amino-modified single-stranded DNA (ssDNA). After hybridization with fluorescently labeled ssDNA, the fluorescence microscopy characterization reveals that: (i) PDA can be toposelectively deposited at the microscale and (ii) electrochemically deposited PDA can be functionalized with amino-terminated ssDNA using the same chemistry as that for spontaneously deposited PDA. Finally, the application of electrodeposited PDA thin films to fabricate ssDNA microarrays is reported using SPR imaging (SPRI) measurements for the detection of DNA and DNA-modified gold nanoparticles.

Fabrication of DNA Microarrays on Polydopamine-Modified Gold Thin Films for SPR Imaging Measurements

Polydopamine (PDA) films were fabricated on thin film gold substrates in a single-step polymerization-deposition process from dopamine solutions and then employed in the construction of robust DNA microarrays for the ultrasensitive detection of biomolecules with nanoparticle-enhanced surface plasmon resonance (SPR) imaging. PDA multilayers with thicknesses varying from 1 to 5 nm were characterized with a combination of scanning angle SPR and AFM experiments, and 1.3 ± 0.2 nm PDA multilayers were chosen as an optimal thickness for the SPR imaging measurements. DNA microarrays were then fabricated by the reaction of amine-functionalized single-stranded DNA (ssDNA) oligonucleotides with PDA-modified gold thin film microarray elements, and were subsequently employed in SPR imaging measurements of DNA hybridization adsorption and protein-DNA binding. Concurrent control experiments with non-complementary ssDNA sequences demonstrated that the adhesive PDA multilayer was also able to provide good resistance to the nonspecific binding of biomolecules. Finally, a series of SPR imaging measurements of the hybridization adsorption of DNA-modified gold nanoparticles onto mixed sequence DNA microarrays were used to confirm that the use of PDA multilayer films is a simple, rapid, and versatile method for fabricating DNA microarrays for ultrasensitive nanoparticle-enhanced SPR imaging biosensing.

Characterization of Electrodeposited Gold and Palladium Nanowire Gratings with Optical Diffraction Measurements

Parallel arrays of either Au or Pd nanowires were fabricated on glass substrates via the electrochemical process of lithographically patterned nanowire electrodeposition (LPNE) and then characterized with scanning electron microscopy (SEM) and a series of optical diffraction measurements at 633 nm. Nanowires with widths varying from 25 to 150 nm were electrodeposited onto nanoscale Ni surfaces created by the undercut etching of a photoresist pattern on a planar substrate. With the use of a simple transmission grating geometry, up to 60 diffraction orders were observed from the nanowire gratings, with separate oscillatory intensity patterns appearing in the even and odd diffraction orders. The presence of these intensity oscillations is attributed to the LPNE array fabrication process, which creates arrays with alternating interwire spacings of distances d +Delta and d -Delta, where d = 25 microm and the asymmetry Delta varied from 0 to 3.5 microm. The amount of asymmetry could be controlled by varying the LPNE undercut etching time during the creation of the nanoscale Ni surfaces. The Fourier transform of a mathematical model of the nanowire array was used to predict the diffraction intensity patterns and quantitatively determine Delta for any grating. Additional sensitivity and an expanded diffraction order range were obtained through the use of external reflection (ER) and total internal reflection (TIR) diffraction geometries.

Wide-field, surface-sensitive four-wave mixing microscopy of nanostructures

We describe a wide-field four-wave mixing (FWM) microscope with imaging characteristics optimized for examining nanostructures. The microscope employs surface-plasmon polariton (SPP) excitation in a gold film to achieve surface-sensitive imaging conditions. The SPP surface fields boost the FWM efficiency by 2 orders of magnitude relative to the excitation efficiency of the evanescent fields at a bare glass surface. We demonstrate two excitation geometries that completely suppress the electronic FWM response of the metal film while allowing the far-field detection of FWM radiation from nanostructures at the interface. We obtained wide-field FWM images from individual carbon nanotubes and nanoclusters of neocyanine molecules at image acquisition times of 1 s, demonstrating the potential for background free, surface-enhanced FWM imaging of nanomaterials.

Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

Recently developed tissue-hydrogel methods for specimen expansion now enable researchers to perform super-resolution microscopy with ∼65 nm lateral resolution using ordinary microscopes, standard fluorescent probes, and inexpensive reagents. Here we use the combination of specimen expansion and the optical super-resolution microscopy technique structured illumination microscopy (SIM) to extend the spatial resolution to ∼30 nm. We apply this hybrid method, which we call ExSIM, to study the cytoskeleton of the important human pathogen Giardia lamblia including the adhesive disc and flagellar axonemes. We determined the localization of two recently identified disc-associated proteins, including DAP86676 , which localizes to disc microribbons, and the functionally unknown DAP16263 , which primarily localizes to dorsal microtubules of the disc overlap zone and the paraflagellar rod of ventral axonemes. Based on its strong performance in revealing known and unknown details of the ultrastructure of Giardia, we find that ExSIM is a simple, rapid, and powerful super-resolution method for the study of fixed specimens, and it should be broadly applicable to other biological systems of interest.