Activation of d-Asparagine and d-Glutamine Derivatives Using the Mitsunobu Reaction.

Seven d-amino acid derivatives having reactive side chains have been activated to afford their respective 3,5-dinitrobenzyl esters using the Mitsunobu reaction. This esterification was found to be difficult using traditional methods involving 3,5-dinitrobenzyl chloride under alkaline conditions. The conversion of a tRNA to the respective d-glutaminyl-tRNA using d-glutamine 3,5-dinitrobenzyl ester was catalyzed by a flexizyme, followed by purification to remove all the unacylated tRNAs and other byproducts. Both d- and l-glutamine were incorporated from their aminoacyl-tRNAs into a model peptide structurally related to IFN-ß.

Ultrasensitive detection of circulating exosomes with a 3D-nanopatterned microfluidic chip.

The performance of current microfluidic methods for exosome detection is constrained by boundary conditions, as well as fundamental limits to microscale mass transfer and interfacial exosome binding. Here, we show that a microfluidic chip designed with self-assembled three-dimensional herringbone nanopatterns can detect low levels of tumour-associated exosomes in plasma (10 exosomes µl-1, or approximately 200 vesicles per 20 µl of spiked sample) that would otherwise be undetectable by standard microfluidic systems for biosensing. The nanopatterns promote microscale mass transfer, increase surface area and probe density to enhance the efficiency and speed of exosome binding, and permit drainage of the boundary fluid to reduce near-surface hydrodynamic resistance, thus promoting particle-surface interactions for exosome binding. We used the device for the detection-in 2 µl plasma samples from 20 ovarian cancer patients and 10 age-matched controls-of exosome subpopulations expressing CD24, epithelial cell adhesion molecule and folate receptor alpha proteins, and suggest exosomal folate receptor alpha as a potential biomarker for early detection and progression monitoring of ovarian cancer. The nanolithography-free nanopatterned device should facilitate the use of liquid biopsies for cancer diagnosis.

Focused Glycomic Profiling With an Integrated Microfluidic Lectin Barcode System.

Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges associated with the analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and the speed of lectin microarrays. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, the CA125 protein purified from ovarian cancer cell lines, and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated "sample-to-answer" microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glycobiomarker development.

Integrated Microfluidic Lectin Barcode Platform for High-Performance Focused Glycomic Profiling.

Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges in analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and speed of lectin microarray. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, CA125 protein purified from ovarian cancer cell line and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated "sample-to-answer" microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glyco-biomarker development.

Characterization of Self-Assembled Monolayers of Peptide Mimotopes of CD20 Antigen and Their Binding with Rituximab.

CD20, expressed in greater than 90% of B-lymphocytic lymphomas, is a target for antibody therapy. Rituximab is a chimeric therapeutic monoclonal antibody (mAb) against the protein CD20, allowing it to destroy B cells and to treat lymphoma, leukemia, transplant rejection, and autoimmune disorder. In this work, the binding of rituximab to self-assembled monolayers (SAMs) of peptide mimotopes of CD20 antigen was systematically characterized. Four peptide mimotopes of CD 20 antigen were selected from the literature and redesigned to allow their SAM immobilizations on gold electrodes through a peptide linker with cysteine. The bindings of these peptides with rituximab and control mAbs (trastuzumab and bevacizumab) were characterized by quartz crystal microbalance (QCM). Among the four peptide mimotopes initially selected, the peptide designated as CN-14 (CGSGSGSWPRWLEN) was the most selective and sensitive for rituximab binding. The CN-14 SAM was further characterized by ellipsometry and atomic force microscopy. The thickness of the CN-14 SAM film was approximately 32 Å, and the CN-14 SAM is suggested to be stabilized by a salt bridge of Arg-10 and Glu-13 between CN-14 peptides. The CN-14 salt bridge was evaluated by a series of modifications to the CN-14 peptide sequence and characterized by QCM. The CN-14 amide variant produced a better affinity to rituximab than CN-14 without a significant impact on selectivity. As the pKa of the Glu residue of CN-14 increased, the affinity of the SAM to rituximab increased, whereas the selectivity decreased. This was attributed to the weakening of the salt bridge between the CN-14 Arg-10 and Glu-13 at higher pKa values for Glu-13. Our study shows that peptide mimotopes have potential benefits in sensor applications, as the peptide-peptide interactions in the SAMs can be manipulated by the addition of functional groups to the peptide to influence the binding of target proteins.

Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35-220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV-vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10(13) M(-2). The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.

Immobilization of a human epidermal growth factor receptor 2 mimotope-derived synthetic peptide on Au and its potential application for detection of herceptin in human serum by quartz crystal microbalance.

Therapeutic antibodies are antigenically similar to human antibodies and are difficult to detect in assays of human serum samples without the use of the therapeutic antibody's complementary antigen. Herein for the first time, we established a platform to detect Herceptin in solutions by using a small (<2.2 kDa), inexpensive, highly stable human epidermal growth factor receptor (HER2) mimotope-derived synthetic peptide immobilized on the surface of a Au quartz electrode. We used the HER2 mimotope as a substitute for the HER2 receptor protein in piezoimmunosensor or quartz crystal microbalance (QCM) assays to detect Herceptin in human serum. We demonstrated that assay sensitivity was dependent upon the amino acids used to tether and link the peptide to the sensor surface and the buffers used to carry out the assays. The detection limit of the piezoimmunosensor assay was 0.038 nM with a linear operating range of 0.038-0.859 nM. Little nonspecific binding to other therapeutic antibodies (Avastin and Rituxan) was observed. Levels of Herceptin in serum samples obtained from treated patients, as ascertained using the synthetic peptide-based QCM assay, were typical for those treated with Herceptin. The findings of this study are significant in that low-cost synthetic peptides could be used in a QCM assay, in lieu of native or recombinant antigens or capture antibodies, to rapidly detect a therapeutic antibody in human serum. The results suggested that a synthetic peptide bearing a particular functional sequence could be applied for developing a new generation of affinity-based immunosensors to detect a broad range of clinical biomarkers.