Halina D. Inerowicz

Spinning-disk self-referencing interferometry of antigen–antibody recognition

A gold ridge microstructure fabricated to a height of lambda/8 on a high-reflectivity substrate behaves as a wave-front-splitting self-referencing interferometer in phase quadrature when illuminated by a Gaussian laser beam and observed in the far field along the optic axis. When immuno-gammaglobulin (IgG) antibodies are selectively immobilized on the gold microstructure, they recognize and bind to a specific antigen, which shifts the relative optical phase of the interferometer and modifies the far-field diffracted intensity. We detect bound antigen interferometrically on spinning disks at a sampling rate of 100 kHz and verify the interferometric nature of the signal by using two quadratures of opposite sign to rule out effects of dynamic light scattering. Strong molecular recognition is demonstrated by the absence of binding to nontarget molecules but strong signal change in response to a specific antigen. This BioCD has the potential to be applied as a spinning-disk interferometric immunoassay and biosensor.

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers.

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ∼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.

Changes in the Proteome of Langat-Infected Ixodes scapularis ISE6 Cells: Metabolic Pathways Associated with Flavivirus Infection

Background Ticks (Family Ixodidae) transmit a variety of disease causing agents to humans and animals. The tick-borne flaviviruses (TBFs; family Flaviviridae) are a complex of viruses, many of which cause encephalitis and hemorrhagic fever, and represent global threats to human health and biosecurity. Pathogenesis has been well studied in human and animal disease models. Equivalent analyses of tick-flavivirus interactions are limited and represent an area of study that could reveal novel approaches for TBF control. Methodology/Principal Findings High resolution LC-MS/MS was used to analyze the proteome of Ixodes scapularis (Lyme disease tick) embryonic ISE6 cells following infection with Langat virus (LGTV) and identify proteins associated with viral infection and replication. Maximal LGTV infection of cells and determination of peak release of infectious virus, was observed at 36 hours post infection (hpi). Proteins were extracted from ISE6 cells treated with LGTV and non-infectious (UV inactivated) LGTV at 36 hpi and analyzed by mass spectrometry. The Omics Discovery Pipeline (ODP) identified thousands of MS peaks. Protein homology searches against the I. scapularis IscaW1 genome assembly identified a total of 486 proteins that were subsequently assigned to putative functional pathways using searches against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. 266 proteins were differentially expressed following LGTV infection relative to non-infected (mock) cells. Of these, 68 proteins exhibited increased expression and 198 proteins had decreased expression. The majority of the former were classified in the KEGG pathways: “translation”, “amino acid metabolism”, and “protein folding/sorting/degradation”. Finally, Trichostatin A and Oligomycin A increased and decreased LGTV replication in vitro in ISE6 cells, respectively. Conclusions/Significance Proteomic analyses revealed ISE6 proteins that were differentially expressed at the peak of LGTV replication. Proteins with increased expression following infection were associated with cellular metabolic pathways and glutaminolysis. In vitro assays using small molecules implicate malate dehydrogenase (MDH2), the citrate cycle, cellular acetylation, and electron transport chain processes in viral replication. Proteins were identified that may be required for TBF infection of ISE6 cells. These proteins are candidates for functional studies and targets for the development of transmission-blocking vaccines and drugs.

Structural Characterization of Plasmenylcholine Photooxidation Products

Oxidative damage to plasmenyl‐type lipids contributes to decreased membrane barrier function, loss of membrane structure and formation of nonlamellar defects in membrane bilayers. Previous results from this laboratory have shown that membrane‐soluble sensitizers (e.g. zinc phthalocyanine and bacteriochlorophyll a) mediate the photooxidation of palmitoyl plasmenylcholine (1‐O‐alk‐1′‐Z‐enyl‐2‐palmitoyl‐sn‐glycero‐3‐phosphocholine; PPlsC) vesicles with the subsequent creation of lamellar defect structures, vesicle contents leakage and membrane–membrane fusion. Because plasmalogen lipids are significant components of sarcoplasma and myelin membranes, we sought to characterize the products of their photooxidation. This study focuses on the photooxidation of PPlsC vesicles in the presence of the water‐soluble sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS44−). Attack of photogenerated singlet oxygen on the 1‐O‐alkenyl ether linkage of PPlsC lipids was expected to generate dioxetane‐ and ene‐type photoproducts. The products formed during continuous aerobic irradiation (28 mW/cm2, (610 nm) of PPlsC vesicles in the presence of AlPcS44− were separated via reverse‐phase high‐performance liquid chromatography (HPLC) with electrochemical detection (ECD) or evaporative light‐escattering detection (ELSD). Photooxidized dipalmitoylphosphatidylcholine–cholesterol vesicles (control) were used to optimize the HPLC‐ECD conditions, using 7α‐hydroperoxycholesterol as standard. HPLC‐ECD was found to be most sensitive for PPlsC hydroperoxides, whereas HPLC‐ELSD was more sensitive for nonhydroperoxide photoproducts. The three major photoproducts formed during vesicle irradiation were isolated via preparative HPLC and then characterized by 1H–nuclear magnetic resonance and mass spectrometry. 1‐Formyl‐2‐palmitoyl‐sn‐glycero‐3‐phosphocholine and 1‐hydroxy‐2‐palmitoyl‐sn‐glycero‐3‐phosphocholine were identified as dioxetane cleavage products that coeluted at ∼3 min. The second fraction (retention time [RT]= 48 min) was identified as a PPlsC allylic hydroperoxide. The third photoproduct, eluting at RT= 64 min, is tentatively identified as an oxidation product arising from allylic hydroperoxide degradation via Hock rearrangement or free radical decomposition.

High-speed label-free multianalyte detection through microinterferometry

Interferometers can detect optical path changes down to a billionth-lambda at the half intensity point at quadrature, (defined when the signal and reference waves are out of phase by ninety degrees). We have fabricated interferometric microstructures on silicon all operating at quadrature. The ultimate capability of this approach is the fabrication of over a billion interferometric biosensors on a single spinning disk having the capacity for mega-samples per second sampling speed. As an initial proof of principle of this technique, we have detected the presence of immobilized anti-mouse IgG and the specific binding of mouse IgG at a sampling rate of 100kiloSamp/sec, while non-specific binding observed was low. We will demonstrate that this technique provides a label-free method that may rapidly screen thousands of proteins per assay.

Spinning-disk laser interferometers for immuno-assays and proteomics: the BioCD

Spinning-disk self-referencing laser interferometers are being developed as high-speed high-sensitivity platforms for immunoassay and proteomics applications. Their compact disc (CD) formats have the potential for ultra-high-throughput multianalyte assays as well as for binding kinetics and quantitative analysis. Self-referencing interferometers are immune to mechanical variations, enabling interferometric sensitivities and speeds that are several orders of magnitude larger than for their counterpart fluorometric techniques. This paper defines for the first time three classes of the BioCD that differ in their method of self-referencing and reviews their relative merits and sensitivities. Each uses a near-field probe with far-field detection. The three classes are: microdiffraction, adaptive optical, and photonic cavity.

Multi-analyte array microdiffraction interferometry

Interferometers have maximum linear sensitivity to small optical perturbations at the half intensity point defined by quadrature when the signal and reference waves are out of phase by ninety degrees. Under this conditions optical path changes down to a billionth-lambda can be detected. We describe the fabrication of interferometric microstructures on silicon all operating at quadrature, which can have sub- micron dimensions. These dimensions could enable, in principle, the fabrication of over a billion interferometer elements on a disk the size of a CD. In addition, a spinning disk of these elements could have the capacity for mega- samples per second sampling rate. As a step towards that goal, we fabricated a 3-inch silicon wafer with 1024 micro strips of gold, each 20 micro strips of gold, each 20 microns wide and 3 cm long arranged radially. The gold was evaporated to a thickness of 79.1 nm putting them at quadrature for read-out with a 632.8 nm He-Ne laser. Bovine Serium Albumin (BSA), was immobilized on the gold micro strips on half of the wafer which had been treated with Hexadecanethiol. The presence of immobilized BSA modified the lobe structure of the far-field diffraction from the micro strips, we are currently extending these static detection result to high-sampling rates on a spinning disk.

Real-time spinning-disk interferometric immunoassays

We describe real-time detection of antigen/antibody binding using a high-speed label-free interferometric detection technique. The sensor, called the BioCD, consists of microfabricated gold interferometric structures on 2” dielectric laser mirror substrates that spin at rates up to 6000 rpm. The interferometric microdiffraction elements operate in the linear sensitivity regime of the interferometer. Antibodies or proteins are immobilized on the gold interferometric structures through an intermediate thiol layer. The molecules are immobilized by application of reagents or samples to the disk while it is spinning. The centrifugal force distributes the sample over the sensor surface, causing a change in the optical phase of the interferometric element, which is detected in real-time using lock-in detection with small detection bandwidth. The thiolated BioCD is spun at 1500 rpm and anti-mouse IgG, rabbit IgG and mouse IgG are delivered in succession to the sensor surface with potash buffered saline (PBS) wash cycles interspersed between each exposure to remove excess unbound proteins. The layer of anti-mouse IgG binds to the thiolated gold sensor elements which later bind specific mouse IgG. We have observed a 10% change in the interferometric signal when mouse IgG binds to an immobilized layer of anti-mouse IgG. No significant non-specific binding of rabbit IgG was detected. The sensitivity and throughput of this sensor will be discussed. An advantage of this new approach, relative to previous work in which the disk was incubated with antigen off-line, is the real-time detection of antigen binding, which could be valuable for simultaneous high-speed screening of a large number of protein interactions.

Photonic phase control in a spinning-disk interferometer for high-speed immunoassays

We have been developing the broad-based concept of spinning-disk interferometry for high-speed multianalyte bioassays with the potential to test up to a thousand specific targets on a disk the size of an audio-CD. The operating principle of spinning-disk interferometry is diffraction off microstructured substrates to achieve phase quadrature between a local reference wave and a sample beam passing through a biolayer. We are currently using this technique to analyze amplitude and phase modulation from immobilized biolayers to determine the degree to which amplitude and phase trade off as single polarizable molecules coalesce into macroscopic dielectric films. We are also studying phase effects associated with the edge of the photonic stop-band that may allow direct quadrature detection of biomolecular films without requiring microstructuring of the substrate.

Protein Microarrays Fabricated by Microcontact Printing

Immunoassays were performed using protein microarrays fabricated by microcontact printing (µCP) techniques. The developed immunoassays were studied by fluorescence microscopy (FM) and scanning probe microscopy (SPM).