Po-Chiao Lin

Fabrication of Oriented Antibody-Conjugated Magnetic Nanoprobes and Their Immunoaffinity Application

In an attempt to fabricate highly active immunoprobes for serum biomarker detection, we report a simple and effective method for site-specific and self-oriented immobilization of antibodies on magnetic nanoparticles (MNPs). Through boronate formation, the carbohydrate moiety within the constant domain, Fc, of the antibody can be specifically and covalently linked to a boronic acid-functionalized MNP (BA@MNP) without hindering the antigen binding domain, Fab. The performance was evaluated by immunoaffinity extraction of multiple serum antigens. Compared with the random immobilization of antibody on a MNP, the antibody self-oriented immunoprobe provides long-term stability (>2 months) and 5-fold extraction efficiency. It also provides 5-fold improved sensitivity at a low nM range (0.4 nM), presumably through enhanced antibody@MNP activity. In addition, false-positive detections arising from nonspecific binding can be completely minimized by effective surface protection using concentration-dependent dextran blocking. Compared with conventional antibody site-specific immobilization through protein G, this new BA-mediated covalent antibody immobilization provides interference-free extraction resulting from noncovalent immobilization of antibody by protein G. The new immunoassay was applied in comparative profiling of serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP) in human serum. Our triple immunoassay revealed a distinct pattern among normal patients, patients with cancer, and patients with cardiovascular disease. Using the previously reported quantization capability of the MALDI MS readout, we expect that this site-specific immunonanoprobe-based immunoassay can be highly active, rapid, and accurate in nanodiagnosis.

Multiplexed Immunoassay: Quantitation and Profiling of Serum Biomarkers Using Magnetic Nanoprobes and MALDI-TOF MS

Taking advantage of efficient affinity extraction by surface-functionalized magnetic nanoparticles (MNPs) and accurate MALDI-TOF MS readout, we present a multiplexed immunoassay for simultaneous enrichment and quantitation of multiple disease-associated antigens, serum amyloid A (SAA), C-reactive protein (CRP), and serum amyloid P (SAP) from human serum. To obtain reproducible MALDI signal response with direct on-MNP detection, the seed-layer method improved homogeneity of the cocrystallization of MNPs and captured antigens. Our methodology demonstrated good quantitation linearity of targeted analytes (R(2) approximately 0.97) with reduced signal variation (RSD < 10%). The lower limit of quantitation is in the nanogram level with overall assay precision (intraday, 7.0%; interday, 11.3%) and accuracy (intraday, 6.3%; interday, 17.5%) including steps of nanoprobe extraction and MALDI-TOF MS analysis. This triplexed immunoassay showed overexpression of SAA and CRP in patients with cardiac catheterization or gastric cancer (P < 0.05), consistent with single-analyte ELISA and previous studies. Compared to the determination of disease onset by single protein quantitation, our multiplexed immunoassay revealed a distinct triplexed pattern in the control group, patients with gastric cancer, and cardiac catheterization. On the basis of the advantages of flexibility in nanoprobe preparation, high specificity and sensitivity, and rapid screening by MALDI-TOF MS, this platform may provide a new methodology for disease diagnosis.

Dihydrobenzoic acid modified nanoparticle as a MALDI-TOF MS matrix for soft ionization and structure determination of small molecules with diverse structures

Efficient structural characterization is important for quality control when developing novel materials. In this study, we demonstrated the soft ionization capability of the hybrid of immobilized silica and 2,5-dihydrobenzoic acid (DHB) on iron oxide magnetic nanoparticles in MALDI-TOF MS with a clean background. The ratio between SiO2 and DHB was examined and was found to affect the surface immobilization of DHB on the nanoparticle, critically controlling the ionization efficiency and interference background. Compared with commercial DHB, the functionalized nanoparticle-assisted MALDI-TOF MS provided superior soft ionization with production of strong molecular ions within 5 ppm mass accuracy on a variety of new types of synthetic materials used for solar cells, light emitting devices, dendrimers, and glycolipids, including analytes with either thermally labile structures or poor protonation tendencies. In addition, the enhancements of the molecular ion signal also provided high-quality product-ion spectra allowing structural characterization and unambiguous small molecule identification. Using this technique, the structural differences among the isomers were distinguished through their characteristic fragment ions and comprehensive fragmentation patterns. With the advantages of long-term stability and simple sample preparation by deposition on a regular sample plate, the use of DHB-functionalized nanoparticles combined with high-resolution MALDI-TOF MS provides a generic platform for rapid and unambiguous structure determination of small molecules.

Site-specific immobilization of CMP-sialic acid synthetase on magnetic nanoparticles and its use in the synthesis of CMP-sialic acid

Through the native chemical ligation, CMP-sialic acid synthetase (CSS) was site-specifically immobilized on magnetic nanoparticles and presented excellent enzymatic performance.

Nanomaterial Based Affinity Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Biomolecules and Pathogenic Bacteria

This paper describes mass spectrometry (MS) using nanoparticles (NPs) for the analysis of biomolecules such as aminothiol compounds, drugs, peptides, proteins, and bacteria. Papers and patents dealing with preparation and use of several NPs in MS have been briefly reviewed, including carbon nanotubes, gold NPs, and magnetic NPs. The NPs or bioconjugated NPs were used for selective concentration and/or assisted matrices for desorption and ionization of analytes of interest. When compared to conventional organic matrixes, the NPs provide low MS background in low-mass region and low shot-to-shot variation. MS techniques using NPs and bioconjugated NPs for the analysis of disease-associated biomarkers and bacteria in real samples such as blood and urine are highlighted, showing the advantages of high sensitivity, reproducibility, and simplicity.

On chip sorting of bacterial cells using sugar-encapsulated magnetic nanoparticles

Here we describe an integrated microfluidic sorting device, which utilized sugar-encapsulated magnetic nanoparticles to separate a specific strain of bacteria from a mixture solution. In our system, microfluidic devices consisting of two inlets and an electromagnet or a permanent magnet have been constructed by a soft lithography process. The magnetic field generated by either the electromagnet or the permanent magnet was strong enough to attract the bacteria bound to magnetic nanoparticles to cross the stream boundary of the laminar flow. The sorting efficiency was found to depend on both the flow rate and the strength of the magnetic field. The maximum sorting efficiency was measured to be higher than 90% and the selectivity was almost 100%. Since this microfluidic sorting device was able to separate 103 bacterial cells within 1min, it could be used for pathogenic diagnose applications.

Iron Oxide Nanomatrix Facilitating Metal Ionization in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

The significance and epidemiological effects of metals to life necessitate the development of direct, efficient, and rapid method of analysis. Taking advantage of its simple, fast, and high-throughput features, we present a novel approach to metal ion detection by matrix-functionalized magnetic nanoparticle (matrix@MNP)-assisted MALDI-MS. Utilizing 21 biologically and environmentally relevant metal ion solutions, the performance of core and matrix@MNP against conventional matrixes in MALDI-MS and laser desorption ionization (LDI) MS were systemically tested to evaluate the versatility of matrix@MNP as ionization element. The matrix@MNPs provided 20- to >100-fold enhancement on detection sensitivity of metal ions and unambiguous identification through characteristic isotope patterns and accurate mass (<5 ppm), which may be attributed to its multifunctional role as metal chelator, preconcentrator, absorber, and reservoir of energy. Together with the comparison on the ionization behaviors of various metals having different ionization potentials (IP), we formulated a metal ionization mechanism model, alluding to the role of exciton pooling in matrix@MNP-assisted MALDI-MS. Moreover, the detection of Cu in spiked tap water demonstrated the practicability of this new approach as an efficient and direct alternative tool for fast, sensitive, and accurate determination of trace metal ions in real samples.

DAST-Mediated Regioselective Anomeric Group Migration in Saccharides

When saccharides bearing a sulfur, selenium, or oxygen substituent at the anomeric center and an unprotected hydroxyl group either at C-4 or C-6 were subjected to fluorination with DAST in dichloromethane, a regioselective migration of the anomeric substituent to the C-4 or C-6 position was observed. Certain saccharides gave a mixture of migration and normal fluorination products whereas others yielded mainly or exclusively migration products (beta-glycosyl fluorides). The high thermal and chemical stability of migrated glycosyl fluorides were demonstrated to be an important precursor for many significant carbohydrate analogies. It is therefore suggested that these migrations may have useful applications in organic synthesis.

Fabrication and Applications of Glyconanomaterials

Nanomaterials have unique optical, electronic, or magnetic properties, thus explaining their potential applications in complex biosystems when coupled with biomolecules, such as DNA, peptides, proteins, or carbohydrates. With a large surface-to-volume ratio and homogeneity in aqueous solutions, various biomolecule-conjugated nanomaterials are exploited for elucidating biological interactions. During the past decade, biomolecule-conjugated nanoparticles (NPs) have been prepared and used in diagnostics [1], creative therapeutics [2], biomolecular interactions [3], and in vivo cell imaging [4, 5]. For example, Mirkin et al. developed an ultrasensitive bio-barcode detection method based on oligonucleotide-conjugated gold NP (AuNP) for biomarkers in small amounts in complex biofluids [6]. Weissleder et al. fabricated antibody-conjugated iron oxide NPs and used them to enhance T2 signals in magnetic resonance imaging [7]. Since they have unique magnetic properties, diverse functionalized magnetic nanoparticles (MNPs) have been designed and prepared to purify target proteins from crude cell lysate by simple magnetic separation. Recently, the authors combined antibody-conjugated MNP with matrix-assisted laser desorption/ionization–time of flight (MALDI–TOF) mass spectrometry (MS) as a rapid and cost-effective detection method for diagnosing disease markers in human sera [8–11]. Biomolecule-modified quantum dots (QDs) have also been demonstrated as having promising applications in in vivo imaging, including cell trafficking and targeting. Besides metallic NPs, carbon nanotubes (CNTs) have also been demonstrated to be powerful carriers and to be useful in biological systems because of their high surface utilization efficiency and good size uniformity.

High quality YBa2Cu3O7−δ films with controllable in-plane orientations grown on yttria-stabilized zirconia substrates

The pulsed-laser deposition (PLD) technique was used to grow high TC superconducting YBa2Cu3O7−δ (YBCO) films on both virgin and ion-bombarded yttria-stabilized zirconia (YSZ) substrates. To pattern high TC films for device applications, the ion milling technique is often used to turn virgin YSZ substrates into ion-bombarded substrates. Multilayered processes require the growth of high TC films on these ion-bombarded substrates. The purpose of this work was to investigate the growing conditions for these two kinds of substrate surfaces. We found that high quality 0° in-plane orientation films can be grown on either substrate when the growth temperature is about 810°C. The thin film grown at this temperature has TC of about 90.3K and JC of about 4×106Acm−2 at 77K. On virgin substrates, the in-plane orientations of YBCO films grown within the temperature range of 790–730°C exhibit a mixture of 0° and 45° domains. As the growth temperature decreases, the dominant orientation shifts gradually from 0° to 45°. ...