Design, characterization, and performance of a hard x-ray transmission microscope at the National Synchrotron Light Source II 18-ID beamline.

A transmission X-ray microscope has been designed and commissioned at the 18-ID Full-field X-ray Imaging beamline at the National Synchrotron Light Source II. This instrument operates in the 5-11 keV range, and, with the current set of optics, is capable of 30 nm spatial resolution imaging, with a field of view of about 40 µm. For absorption contrast, the minimum exposure time for a single projection image is about 20 ms and an entire 3D tomography data set can be acquired in under 1 min. The system enables tomographic reconstructions with sub-50 nm spatial resolution without the use of markers on the sample or corrections for rotation run-outs.

In situ fabrication of cleavable peptide arrays on polydimethylsiloxane and applications for kinase activity assays.

Polydimethylsiloxane (PDMS) is widely used for microfabrication and bioanalysis; however, its surface functionalization is limited due to the lack of active functional groups and incompatibility with many solvents. We presented a novel approach for in situ fabrication of cleavable peptide arrays on polydimethylsiloxane (PDMS) viatert-butyloxycarbonyl (t-Boc)/trifluoroacetic acid (TFA) chemistry using gold nanoparticles (AuNPs) as the anchor and a disulfide/amine terminated hetero-polyethylene glycol as the cleavable linker. The method was fine tuned to use reagents compatible with the PDMS. Using 5-mer pentapeptide, Trp5, as a model, step-by-step covalent coupling during the reaction cycles was monitored by Attenuated total reflectance-Fourier transform infrared spectrometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), or atomic force microscopy (AFM), and further confirmed by mass spectrometry (MS) detection of the cleaved peptides. Using such a method, heptapeptides of the PKA substrate, LRRASLG (Kemptide), and its point mutated analogs were fabricated in an array format for comparative studies of cAMP-dependent protein kinase (PKA) activity. Based on on-chip detection, Kemptide sequence exhibited the highest phosphorylation activity, which was detected to a 1.5-time lesser extent for the point mutated sequence (LRRGSLG) containing the recognition motif (RRXS), and was nearly undetectable for another point mutated sequence (LRLASLG) that lacked the recognition motif. These results indicate that the reported fabrication method is able to yield highly specific peptide sequences on PDMS, leading to a highly motif-sensitive enzyme activity assay.

A compact synchrotron-based transmission X-ray microscope.

A compact transmission X-ray microscope has been designed and implemented based on a cylindrical symmetry around the optical axis that sharply limits the instabilities due to thermal mechanical drift. Identical compact multi-axis closed-loop actuation modules drive different optical components. The design is modular and simplifies the change of individual parts, e.g. the use of different magnification and focusing devices. This compact instrument can be easily transported between laboratory and synchrotron facilities and quickly put into operation. An automated alignment mechanism simplifies the assembly of different modules after transportation. After describing the design details, the results of the first tests are presented.

Design and characterization of a compact nano-positioning system for a portable transmission x-ray microscope.

We have designed and constructed a compact nano-positioning system for a Portable Transmission X-ray Microscope (PTXM). We introduce a concept of PTXM and adopt modular approach which implements identical nano-motion platforms to perform manipulation of PTXM components. Modular design provides higher stiffness of the system and allows for reduction of relative thermal drifts between individual constituents of the PTXM apparatus, ensuring a high degree of stability for nanoscale x-ray imaging. We have measured relative thermal drifts between two identical modules to be as low as 15 nm/h, sufficient to perform nanoscale imaging by TXM. Spatial resolution achieved by developed linear piezo stages was measured to be 3 nm with repeatability of 20 nm over 1 mm travel range.

Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays.

The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R(2) > 0.98, indicating the great promise of the developed method for clinical applications.

Functional fluorinated modifications on a polyelectrolyte coated polydimethylsiloxane substrate for fabricating antibody microarrays.

Fluorinated compounds exhibit hydrophobic, nonstick, and self-cleaning properties, making them attractive for use as the coating material for biochips. In this study, we copolymerized the fluorinated compound 1H,1H,2H-perfluoro-1-decene (FD) with acrylic acid (AA) and bonded the resulting copolymer with protein G on the surface of polyelectrolyte coated polydimethylsiloxane (PDMS) to form a functional surface that captures antibodies. We demonstrated that the modified PDMS surface remained hydrophobic while becoming resistant to nonspecific protein binding. Thus, aqueous sample solutions formed the droplets (4 µL) on the surface without spreading and drying during the sample printing. Contact angle measurements showed that this functionalized surface was as hydrophobic as the native PDMS with a virtually constant contact angle over seven days of the study under dried condition at 4 °C. Spectroscopic measurements revealed that FD/AA copolymerization formed a homogeneous and highly dense multilayer (50 mg/mm(2)) with a fluorine coverage of 35.4%. Moreover, protein G was shown to covalently bind to AA molecules on the surface at a binding density of 0.24 µg/mm(2). We demonstrated that the fluorinated coating withstood nonspecific binding with extremely low background emission, leading to bioassays that, without the need of blocking agents, exhibited six times more sensitivity than PEG coatings. The fluorinated PDMS antibody microarrays were further applied to accurately determine the absolute concentration of ERα in MCF-7 cells. In conclusion, the unique properties of fluorinated compounds, such as withstanding wetting, nonspecific binding and contamination, make them an excellent coating material for use in sensitive and simple on-chip assays.

Functionalized 3D-hydrogel plugs covalently patterned inside hydrophilic poly(dimethylsiloxane) microchannels for flow-through immunoassays.

Integration of a hydrogel and polydimethylsiloxane (PDMS)-based microfluidic device can greatly reduce the cost of developing channel-based devices. However, there are technical difficulties including the hydrophobic and inert surface properties associated with PDMS as well as back pressure and fragile material associated with the use of hydrogel in microchannels. In this study, a strategy to covalently photopattern 3-D hydrogel plugs with functionalized protein G inside microfluidic channels on a hydrophilic PDMS substrate coated with polyelectrolyte multilayers (PEMS) is presented. In this process, a UV-light microscope is applied to initiate the protein G-poly(acryl amide) copolymerization from the bulk substrate to solution areas via the deeply implanted photoinitiator (PI), resulting in sturdy 3D plugs covalently bonded to the upper and lower channel wall, while leaving open spaces in the channel width for the fluid to flow through. In addition, the long-term hydrophilicity and low nonspecific binding property associated with PEMS surface can be conserved for the nonpatterned area, leading to hydrogel plugs in extremely hydrophilic and permeable environment in a restricted channel space for bubble-free fluid transport and affinity interaction. By immobilization of well-oriented antibodies via protein G on the hydrogel plugs in the channel, estrogen receptor alpha (ERalpha) is demonstrated to be captured quantitatively with high loading capacity and high specificity.

Photopatterning of tough single-walled carbon nanotube composites in microfluidic channels and their application in gel-free separations.

We report on the photopatterning of single carbon nanotube composites with soft hydrogel polymers in glass microchannels. Since the hydrogels by themselves are able to withstand liquid flow within the microchannels, we covalently combined them with single-walled carbon nanotubes to impart mechanical strength. We attempted this approach by patterning the gels within the microchannels without prior surface modifications. Our results show that the 1-cm nanocomposite hydrogels are far stronger than the free hydrogels. Moreover, the nanocomposites were able to concentrate and separate proteins within a 1.5-cm distance using gel-free buffers. The separation cannot only be tuned by changing the running buffer; the lack of gels in the running buffer reduces the chance of channel blockage and thus the lifetime of the device is prolonged. The usefulness of the patterned nanocomposites may be extended by a wide selection of nanocomposite properties and monomers to find a broad range of applications in lab-on-chip technology.