Heterobifunctional Cross-linker Research Articles

Specific Targeting of HER2-Positive Head and Neck Squamous Cell Carcinoma Line HN5 by Idarubicin-ZHER2 Affibody Conjugate.

Expression of human epidermal growth factor receptor type 2 (HER2) in head and neck squamous cell carcinoma (HNSCC) cell line HN5 can be employed with great opportunities of success for specific targeting of anti-cancer chemotherapeutic agents. In the current study, HER2-specific affibody molecule, ZHER2:342 (an engineered protein with great affinity for HER2 receptors) was selected for conjugation to idarubicin (an anti-neoplastic antibiotic). ZHER2:342 affibody gene with one added cysteine code at the its 5' end was synthesized de novo and then inserted into pET302 plasmid and transferred to E. Coli BL21 hosting system. After induction of protein expression, the recombinant ZHER2 affibody molecules were purified using Ni- NTA resin and purity was analyzed through SDS-PAGE. Affinity-purified affibody molecules were conjugated to idarubicin through a heterobifunctional crosslinker, sulfosuccinimidyl 4-(Nmaleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC). Specific toxicity of idarubicin-ZHER2 affibody conjugate against two HER2-positive cells, HN5 and MCF-7 was assessed through MTT assay after an exposure time of 48 hours with different concentrations of conjugate. Idarubicin in the non-conjugated form showed potent toxic effects against both cell lines, while HN5 cells were significantly more sensitive compared to MCF-7 cells. Dimeric ZHER2 affibody showed a mild decreasing effect on growth of both HN5 and MCF-7 cells at optimum concentration. Idarubicin-ZHER2 affibody conjugate at an optimum concentration reduced viability of HN5 cell line more efficiently compared to MCF-7 cell line. In conclusion, idarubicin-ZHER2 affibody conjugate in optimum concentrations can be used for specific targeting and killing of HN5 cells.

Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a "plant-and-cast" cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues "planting" the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then "cast" across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximity-enhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical cross-linking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme-substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.

Synthesis and Characterization of Cetuximab-Docetaxel and Panitumumab-Docetaxel Antibody-Drug Conjugates for EGFR-Overexpressing Cancer Therapy.

The safety and efficacy of anticancer antibody-drug conjugates (ADCs) depend on the selection of tumor-targeting monoclonal antibody (mAb), linker, and drug, as well as their specific chemical arrangement and linkage chemistry. In this study, we used a heterobifunctional cross-linker to conjugate docetaxel (DX) to cetuximab (CET) or panitumumab (PAN). The resulting ADCs were investigated for their in vitro EGFR-specific cytotoxicity and in vivo anticancer activity. Reaction conditions, such as reducing agent, time, temperature, and alkylation buffer, were optimized to yield potent and stable ADCs with consistent batch-to-batch drug-to-antibody ratios (DARs). ADCs were synthesized with DARs from 0.4 to 3.0, and all retained their EGFR affinity and specificity after modification. ADCs were sensitive to cell surface wildtype EGFR expression, demonstrating more cytotoxicity in EGFR-expressing A431 and MDA-MB-231 cell lines compared to U87MG cells. A431 tumor-bearing mice treated once weekly for four weeks with 100 mg/kg cetuximab-docetaxel ADC (C-SC-DX, DAR 2.5) showed durable anticancer responses and improved overall survival compared to the same treatment regimen with 1 mg/kg DX, 100 mg/kg CET, or a combination 1 mg/kg DX and 100 mg/kg CET. New treatment options are emerging for patients with both wild-type and mutated EGFR-overexpressing cancers, and these studies highlight the potential role of EGFR-targeted ADC therapies as a promising new treatment option.

Lysosomal Targeting Enhancement by Conjugation of Glycopeptides Containing Mannose-6-phosphate Glycans Derived from Glyco-engineered Yeast

Many therapeutic enzymes for lysosomal storage diseases require a high content of mannose-6-phosphate (M6P) glycan, which is important for cellular uptake and lysosomal targeting. We constructed glyco-engineered yeast harboring a high content of mannosylphosphorylated glycans, which can be converted to M6P glycans by uncapping of the outer mannose residue. In this study, the cell wall of this yeast was employed as a natural M6P glycan source for conjugation to therapeutic enzymes. The extracted cell wall mannoproteins were digested by pronase to generate short glycopeptides, which were further elaborated by uncapping and α(1,2)-mannosidase digestion steps. The resulting glycopeptides containing M6P glycans (M6PgPs) showed proper cellular uptake and lysosome targeting. The purified M6PgPs were successfully conjugated to a recombinant acid α-glucosidase (rGAA), used for the treatment of Pompe disease, by two-step reactions using two hetero-bifunctional crosslinkers. First, rGAA and M6PgPs were modified with crosslinkers containing azide and dibenzocyclooctyne, respectively. In the second reaction using copper-free click chemistry, the azide-functionalized rGAA was conjugated with dibenzocyclooctyne-functionalized M6PgPs without the loss of enzyme activity. The M6PgP-conjugated rGAA had a 16-fold higher content of M6P glycan than rGAA, which resulted in greatly increased cellular uptake and efficient digestion of glycogen accumulated in Pompe disease patient fibroblasts.

Quantitative Analysis of Peptide-Matrix Interactions in Lyophilized Solids Using Photolytic Labeling.

Peptide-matrix interactions in lyophilized solids were explored using photolytic labeling with reversed phase high performance liquid chromatography (rp-HPLC) and mass spectrometric (MS) analysis. A model peptide (Ac-QELHKLQ-NHCH3) derived from salmon calcitonin was first labeled with a heterobifunctional cross-linker NHS-diazirine (succinimidyl 4,4'-azipentanoate; SDA) at Lys5 in solution, with ∼100% conversion. The SDA labeled peptide was then formulated with the following excipients at a 1:400 molar ratio and lyophilized: sucrose, trehalose, mannitol, histidine, arginine, urea, and NaCl. The lyophilized samples and corresponding solution controls were exposed to UV at 365 nm to induce photolytic labeling, and the products were identified by MS and quantified with rp-HPLC or MS. Peptide-excipient adducts were detected in the lyophilized solids except the NaCl formulation. With the exception of the histidine formulation, peptide-excipient adducts were not detected in solution and the fractional conversion to peptide-water adducts in solution was significantly greater than in lyophilized solids, as expected. In lyophilized solids, the fractional conversion to peptide-water adducts was poorly correlated with bulk moisture content, suggesting that the local water content near the labeled lysine residue differs from the measured bulk average. In lyophilized solids, the fractional conversion to peptide-excipient adducts was assessed using MS extracted ion chromatograms (EIC); subject to the assumption of equal ionization efficiencies, the fractional conversion to excipient adducts varied with excipient type. The results demonstrate that the local environment near the lysine residue of the peptide in the lyophilized solids can be quantitatively probed with a photolytic labeling method.

Layer-by-Layer Heparinization of the Cell Surface by Using Heparin-Binding Peptide Functionalized Human Serum Albumin.

Layer-by-layer heparinization of therapeutic cells prior to transplantation is an effective way to inhibit the instant blood-mediated inflammatory reactions (IBMIRs), which are the major cause of early cell graft loss during post-transplantation. Here, a conjugate of heparin-binding peptide (HBP) and human serum albumin (HSA), HBP-HSA, was synthesized by using heterobifunctional crosslinker. After the first heparin layer was coated on human umbilical vein endothelial cells (HUVECs) by means of the HBP-polyethylene glycol-phospholipid conjugate, HBP-HSA and heparin were then applied to the cell surface sequentially to form multiple layers. The immobilization and retention of heparin were analyzed by confocal microscopy and flow cytometry, respectively, and the cytotoxity of HBP-HSA was further evaluated by cell viability assay. Results indicated that heparin was successfully introduced to the cell surface in a layer-by-layer way and retained for at least 24 h, while the cytotoxity of HBP-HSA was negligible at the working concentration. Accordingly, this conjugate provides a promising method for co-immobilization of heparin and HSA to the cell surface under physiological conditions with improved biocompatibility.

False discovery rate estimation and heterobifunctional cross-linkers.

False discovery rate (FDR) estimation is a cornerstone of proteomics that has recently been adapted to cross-linking/mass spectrometry. Here we demonstrate that heterobifunctional cross-linkers, while theoretically different from homobifunctional cross-linkers, need not be considered separately in practice. We develop and then evaluate the impact of applying a correct FDR formula for use of heterobifunctional cross-linkers and conclude that there are minimal practical advantages. Hence a single formula can be applied to data generated from the many different non-cleavable cross-linkers.

Functionalisation of Detonation Nanodiamond for Monodispersed, Soluble DNA-Nanodiamond Conjugates Using Mixed Silane Bead-Assisted Sonication Disintegration

Nanodiamonds have many attractive properties that make them suitable for a range of biological applications, but their practical use has been limited because nanodiamond conjugates tend to aggregate in solution during or after functionalisation. Here we demonstrate the production of DNA-detonation nanodiamond (DNA-DND) conjugates with high dispersion and solubility using an ultrasonic, mixed-silanization chemistry protocol based on the in situ Bead-Assisted Sonication Disintegration (BASD) silanization method. We use two silanes to achieve these properties: (1) 3-(trihydroxysilyl)propyl methylphosphonate (THPMP); a negatively charged silane that imparts high zeta potential and solubility in solution; and (2) (3-aminopropyl)triethoxysilane (APTES); a commonly used functional silane that contributes an amino group for subsequent bioconjugation. We target these amino groups for covalent conjugation to thiolated, single-stranded DNA oligomers using the heterobifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The resulting DNA-DND conjugates are the smallest reported to date, as determined by Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). The functionalisation method we describe is versatile and can be used to produce a wide variety of soluble DND-biomolecule conjugates.

ESI-MS and Stavrox 3.6.0.1 Investigations of Crosslinking by an Aryl-Azido-NHS-Heterobifunctional Crosslinker

Chemical cross-linking-mass spectrometry (CX-MS) combined with bioinformatics tools is increasingly being used to analyze large-scale protein–protein interactions. It has gained importance in studies in proteomics, lipidomics, in systems and structural biology. Recently it has gained importance in preparation of homogeneous antibody-drug conjugates, which has been described as “a pinnacle of such targeting efforts.” What makes these approaches exciting is that using the “Click” and Bertozzi protocols in vivo studies can be carried out successfully. Using CX-MS combined with cryo-EM, structures of protein complexes can now be probed at almost molecular resolution (upto 3 Å). Chemical crosslinking is useful in materials science, as well. Major advances in both mass spectrometric techniques and bioinformatics tools today allow one to identify cross-linked peptides with highconfidence and with more user-friendly approaches. Crucial to this is the ability to capture intermolecular crosslinking reliably. The use of a new small NHS-aryl azido heterobifunctional cross-linker based is described here, which picks intermolecular crosslinking better. Thus, Lysozyme has been crosslinked successfully as established by the ‘dimeric’ band observed in SDS-PAGE. its tryptic digestion, ‘zip tip’ enrichment, ESI-MS, MS/MS and the data generated analyzed using StavroX 3.6.0.1, a bioinformatics software, especially suited for determining intermolecular crosslinking.

Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p).

Development and Characterization of a Camelid Single Domain Antibody-Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2.

Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs). In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21) and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody–urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MSE peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the E. coli BL21 (DE3) pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester (SM(PEG)2), which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol (BM(PEG)2), which targets the cysteine added to the antibody C-terminus. V21H4-DOS47 was determined to be the superior conjugate as the antibody is easily produced and purified at high levels, and the conjugate can be efficiently generated and purified using methods easily transferrable for cGMP production. In addition, V21H4-DOS47 retains higher binding activity than V21H1-DOS47, as the native lysine residues are unmodified.

A Facile Way of Modifying Layered Double Hydroxide Nanoparticles with Targeting Ligand-Conjugated Albumin for Enhanced Delivery to Brain Tumour Cells.

Active targeting of nanoparticles (NPs) for cancer treatment has attracted increasing interest in the past decades. Various ligand modification strategies have been used to enhance the targeting of NPs to the tumor site. However, how to reproducibly fabricate diverse targeting NPs with narrowly changeable biophysiochemical properties remains as a major challenge. In this study, layered double hydroxide (LDH) NPs were modified as a target delivery system. Two brain tumor targeting ligands, i.e., angiopep-2 and rabies virus glycoprotein, were conjugated to the LDH NPs via an intermatrix protein moiety, bovine serum albumin (BSA), simultaneously endowing the LDHs with excellent colloidal stability and targeting capability. The ligands were first covalently linked with BSA through the heterobifunctional cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. Then, the ligand-linked BSA and pristine BSA were together coated onto the surface of LDHs through electrostatic interaction, followed by cross-linking with the cross-linker glutaraldehyde to immobilize these BSAs on the LDH surface. In this way, we are able to readily prepare colloidally stabilized tumor-targeted LDH NPs. The targeting efficacy of the ligand-conjugated LDH delivery system has been evidenced in the uptake by two neutral cells (U87 and N2a) compared to unmodified LDHs. This new approach provides a promising strategy for rational design and preparation of target nanoparticles as a selective and effective therapeutic treatment for brain tumors.

Selective targeted delivery of doxorubicin via conjugating to anti-CD24 antibody results in enhanced antitumor potency for hepatocellular carcinoma both in vitro and in vivo.

Antibody-drug conjugates (ADCs) represent a promising therapeutic approach for clinical application. Cluster of differentiation 24 (CD24) is over-expressed in several human malignancies, especially in hepatocellular carcinoma (HCC). We aimed to develop a new class of CD24-targeted ADCs for HCC. DOX was conjugated with G7mAb by a heterobifunctional cross-linker GMBS (N-[gamma-maleimido butyryloxy] succinimide ester) and further analyzed using HPLC. The targeting specificity and endocytosis of the newly generated ADC, G7mAb-DOX, were characterized using flow cytometry assay, near-infrared fluorescence imaging and laser scanning confocal microscope. The antitumor effects were evaluated in nude mice bearing HCC xenografts. G7mAb-DOX with average two drug molecules per antibody was selectively captured and endocytosed by CD24 (+) tumor cells in vitro. In vivo, the ADC was proved to target tumor tissues, suppress tumor growth and prolong the survival of HCC-bearing nude mice with improved efficacy and less systemic toxicity compared with either G7mAb or DOX single-agent treatment. These studies provide proof of concept for development of DOX-based ADCs which provide a novel approach for HCC-targeted immune therapy in clinical application.

A sensitive and disposable electrochemical immunosensor for detection of SOX2, a biomarker of cancer

A novel, sensitive, disposable indium tin oxide (ITO)-based electrochemical immunosensor was developed firstly for simple, rapid determination of Sex-determining region Y-box 2 (SOX2). SOX2 is a cancer biomarker and used for detecting small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. In this study, a disposable ITO thin film based electrode was used as working electrode for biosensing the interaction between SOX2 antigen and anti-SOX2 antibody. In this study, carboxyethylsilanetriol (CTES) was also utilized for electrode modifying so as to obtain self-assembled monolayers. The formed self-assembled monolayers were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) chemistry and they were used as a heterobifunctional crosslinker and activator, respectively. Anti-SOX2 antibody was used as a biorecognition molecule and was covalently immobilized onto the ITO thin film modified with CTES. Immobilization steps were characterized by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The optimum immobilization conditions such as antibody concentration, antibody and antigen incubation times were examined for the best sensitivity of the immunosensor. Under optimal conditions, this immunosensor had a wide linear detection range (25fg/mL–2pg/mL) with a detection limit as low as 7fg/mL SOX2. Furthermore, the developed SOX2 immunosensor had good storage stability (79.36% of initial activity after 9 weeks), repeatability (3.88% of RSD) and reproducibility (4.25% of RSD). Our developed immunosensor has an acceptable performance for detection of SOX2 antigen, exhibits low detection limit, and has selective and reproducible results in immunoreaction analysis.

Complementary Benzophenone Cross-Linking/Mass Spectrometry Photochemistry.

Use of a heterobifunctional photoactivatable cross-linker, sulfo-SDA (diazirine), has yielded high-density data that facilitated structure modeling of individual proteins. We expand the photoactivatable chemistry toolbox here with a second reagent, sulfo-SBP (benzophenone). This further increases the density of photo-cross-linking to a factor of 20× over conventional cross-linking. Importantly, the two different photoactivatable groups display orthogonal directionality, enabling access to different protein regions, unreachable with a single cross-linker.

Fast-responsive hydrogel as an injectable pump for rapid on-demand fluidic flow control.

Chemically synthesized functional hydrogels have been recognized as optimized soft pumps for on-demand fluidic regulation in micro-systems. However, the challenges regarding the slow responses of hydrogels have very much limited their application in effective fluidic flow control. In this study, a heterobifunctional crosslinker (4-hydroxybutyl acrylate)-enabled two-step hydrothermal phase separation process for preparing a highly porous hydrogel with fast response dynamics was investigated for the fabrication of novel microfluidic functional units, such as injectable valves and pumps. The cylinder-shaped hydrogel, with a diameter of 9 cm and a height of 2.5 cm at 25 °C, achieved a size reduction of approximately 70% in less than 30 s after the hydrogels were heated at 40 °C. By incorporating polypyrrole nanoparticles as photothermal transducers, a photo-responsive composite hydrogel was approached and exhibited a remotely triggerable fluidic regulation and pumping ability to generate significant flows, showing on-demand water-in-oil droplet generation by laser switching, whereby the droplet size could be tuned by adjusting the laser intensity and irradiation period with programmable manipulation.

PEGylated neuromedin U-8 shows long-lasting anorectic activity and anti-obesity effect in mice by peripheral administration

Neuromedin U (NMU) is a neuropeptide found in the brain and gastrointestinal tract. The NMU system has been shown to regulate energy homeostasis by both a central and a peripheral mechanism. Peripheral administration of human NMU-25 was recently shown to inhibit food intake in mice. We examined the possibility that other NMU-related peptides exert an anorectic activity by intraperitoneal (i.p.) administration. We found that rat NMU-23 and its structurally-related peptide rat neuromedin S (NMS) significantly reduced food intake in lean mice, whereas NMU-8, an active fragment of the octapeptide sequence conserved in porcine, human and mouse NMU, had no effect. When rat NMU-23, NMU-8, and rat NMS were covalently conjugated to polyethylene glycol (PEG) (PEGylation) at the N-terminus of these peptides, PEGylated NMU-8 showed the most long-lasting and robust anorectic activity. The exploration of the linker between NMU-8 and PEG using hetero-bifunctional chemical cross-linkers led to an identification of PEGylated NMU-8 analogs with higher affinity for NMU receptors and with more potent anorectic activity in lean mice. The PEGylated NMU-8 showed potent and robust anorectic activity and anti-obesity effect in diet-induced obesity (DIO) mice by once-daily subcutaneous (s.c.) administration. These results suggest that PEGylated NMU-8 has the therapeutic potential for treatment of obesity.

Graphene oxide functionalized long period grating for ultrasensitive label-free immunosensing

We explore graphene oxide (GO) nanosheets functionalized dual-peak long period grating (dLPG) based biosensor for ultrasensitive label-free antibody-antigen immunosensing. The GO linking layer provides a remarkable analytical platform for bioaffinity binding interface due to its favorable combination of exceptionally high surface-to-volume ratio and excellent optical and biochemical properties. A new GO deposition technique based on chemical-bonding in conjunction with physical-adsorption was proposed to offer the advantages of a strong bonding between GO and fiber device surface and a homogeneous GO overlay with desirable stability, repeatability and durability. The surface morphology of GO overlay was characterized by Atomic force microscopy, Scanning electron microscope, and Raman spectroscopy. By depositing the GO with a thickness of 49.2nm, the sensitivity in refractive index (RI) of dLPG was increased to 2538nm/RIU, 200% that of non-coated dLPG, in low RI region (1.333–1.347) where bioassays and biological events were usually carried out. The IgG was covalently immobilized on GO-dLPG via EDC/NHS heterobifunctional cross-linking chemistry leaving the binding sites free for target analyte recognition. The performance of immunosensing was evaluated by monitoring the kinetic bioaffinity binding between IgG and specific anti-IgG in real-time. The GO-dLPG based biosensor demonstrates an ultrahigh sensitivity with limit of detection of 7ng/mL, which is 10-fold better than non-coated dLPG biosensor and 100-fold greater than LPG-based immunosensor. Moreover, the reusability of GO-dLPG biosensor has been facilitated by a simple regeneration procedure based on stripping off bound anti-IgG treatment. The proposed ultrasensitive biosensor can be further adapted as biophotonic platform opening up the potential for food safety, environmental monitoring, clinical diagnostics and medical applications.

Electrochemical immunoassay for amyloid-beta 1–42 peptide in biological fluids interfacing with a gold nanoparticle modified carbon surface

An electrochemical immunosensor involving the formation of a surface sandwich complex on a gold nanoparticle (NP) modified screen printed carbon electrode (SPCE) is demonstrated for the femtomolar detection of amyloid-beta 1–42 peptide (Aβ) in both serum and plasma. Both bioreceptors forming the assay are highly selective antibodies for Aβ, namely antiAβ (12F4) and (1E11) which possess different binding sites for the Aβ peptide. In order to improve the sensing performance for complex biological fluidic matrix analysis, different mixed monolayers of thiol modified polyethylene glycol (PEG) and mercaptopropionic acid (MPA) were self-assembled onto the Au NP-SPCE followed by tethering antiAβ (12F4) to MPA using a heterobifunctional cross linker. Surface sandwich complexes of antiAβ (12F4)/Aβ/antiAβ (1E11)-ALP were then formed via sequential adsorption with the latter antiAβ (1E11) conjugated to alkaline phosphatase (ALP) enzyme. The reaction of surface immobilized ALP with the substrate, 4-amino phenyl phosphate (APP), generated voltammetric detection signals that linearly increased as a function of Aβ concentration. Differential pulse voltammetry was applied to establish a lowest detectable concentration of 100 fM of Aβ with a linear response range from 100 fM to 25 pM. Following optimization, the immunoassay platform was applied in diluted human serum and plasma samples to determine the native concentration of Aβ and the results were validated using a commercially available ELISA test.

Impacts of Cross-Linkers on Biological Effects of Mesoporous Silica Nanoparticles.

Chemically synthesized cross-linkers play decisive roles in variable cargos attached to nanoparticles (NPs). Previous studies reported that surface properties, such as the size, charge, and surface chemistry, are particularly important determinants influencing the biological fate and actions of NPs and cells. Recent studies also focused on the relationship of serum proteins with the surface properties of NPs (also called the protein corona), which is recognized as a key factor in determining the cytotoxicity and biodistribution. However, there is concern that cross-linkers conjugated onto NPs might induce undesirable biological effects. Cell responses induced by cross-linkers have not yet been precisely elucidated. Herein, using mesoporous silica nanoparticles (MSNs) the surfaces of which were separately conjugated with four popular heterobifunctional cross-linkers, i.e., N-[α-maleimidoacetoxy]succinimide ester (AMAS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and maleimide poly(ethylene glycol) succinimidyl carboxymethyl ester (MAL-PEG-SCM), we investigated cross-linker-conjugated MSNs to determine whether they can cause cytotoxicity, or enhance reactive oxygen species (ROS) generation, nuclear factor (NF)-κB activation, and p-p38 or p21 protein expressions in RAW264.7 macrophage cells. Furthermore, we also separately conjugated two biomolecules containing TAT peptides and bovine serum albumin (BSA) as model systems to study their cell responses in detail. Finally, in vivo mice studies evaluated the biodistribution and blood assays (biochemistry and complete blood count) of PEG-derivative NPs, and results suggested that TAT peptides caused significant white blood cell (WBC)-related cell and platelet abnormalities, as well as liver and kidney dysfunction compared to BSA when conjugated onto MSNs. The results showed that attention to cross-linkers should be considered an issue in the surface modification of NPs. We anticipate that our results could be helpful in developing biosafety nanomaterials.