A conjoint multi metal-ion iminodiacetic acid monolith microfluidic chip for structural-based protein pre-fractionation.

PDMS-based multichannel microfluidic chip was designed and fabricated in a simple approach using readily available tools. UV-initiated in situ polymerization of poly(2-hydroxy ethyl methacrylate-co-di(ethylene glycol) diacrylate-co-N,N'-diallyl l-tartardiamide) in an Eppendorf tube was achieved within 40 min. This polymerization process was successfully translated to a microfluidic chip format without any further modifications. Iminodiacetic acid was successfully immobilized on aldehyde functional monoliths via Schiff base reaction and confirmed by FT-IR spectroscopy. Four transition metal ions (Co (II), Zn (II), Ni (II), and Cu (II)) were chelated individually on four IDA-monolith microfluidic chips. The conjoint metal-ion monolith microfluidic chip has displayed high permeability (9.40 × 10-13  m2 ) and a porosity of 32.8%. This affinity microfluidic chip has pre-fractioned four human plasma proteins (fibrinogen, immunoglobulin, transferrin, and human serum albumin) based on their surface-exposed histidine surface topography. A protein recovery of approximately 95% (Bradford assay data) was achieved. The multimonolith microchip can be reusable even after three protein adsorption-desorption cycles.

Unraveling a natural protease inhibitor from marine Streptomyces griseoincarnatus HK12 active against Chikungunya virus.

Proteases play an indispensable role in the life cycles of several life-threatening organisms such as the ones causing malaria, cancer and AIDS. A targeted blockade of these enzymes could be an efficient approach for drug modeling against these causative agents. Our study was directed towards the extraction and characterization of a protease inhibitor having activity against Chikungunya virus (CHIKV). A protein-based protease inhibitor (PI) in Streptomyces griseoincarnatus HK12 with anti-viral activity against CHIKV was revealed when screened against two major proteases, papain and trypsin. The PI was efficiently extracted at 60 % ammonium sulfate saturation and purified by ion-exchange chromatography (CM-Sepharose) at 300 mM NaCl elution followed by SDS-PAGE (10 %). The protein was characterized by denaturing SDS-PAGE, reverse zymography, and MALDI-TOF peptide mass fingerprinting. The protein-based PI was studied to have a high molecular weight of 66-70 kDA. The PI was tested to supress the supress cytopathic effects (CPE) exerted by the clinically isolated virus in BHK21 cells. This was used as a measure to determine the antiviral activity. The PI exerted significant effects with an effective concentration calculated as EC50 11.21 µg/mL. The protein was found to be reported as the first of its kind which also stands out to be the first a natural protease inhibitor against the treatment of the chikungunya virus.

Simplification of affinity macroporous monolith microfluidic column synthesis and its ability for protein separation.

A generic multi-component approach was designed to perform simultaneous in situ polymerization and ligand immobilization to develop affinity porous polymer based chromatography resin in a facile mode. This strategy exploits the regioselective ring opening reaction between epoxy group of monomer and native functional group of ligand (i.e. amine) under aqueous condition (pH 9.7). As a proof-of-concept, reaction of iminodiacetic acid (IDA) with allyl glycidyl ether (AGE) in presence of other monomer (HEMA) and crosslinkers (DATD, PDA) for 4 h via thermal initiation process (temperature of 65 °C) was shown. Successful polymerization (both ex situ &in situ) was confirmed by visual observation, surface morphology of the polymer by scanning electron microscope and ligand immobilization by FT-IR analysis. Chelation of the metal-ion i.e. copper (Cu (II)) with IDA in the monolith showed IgG adsorption capacity (27.8 mg/g monolith) over IDA-monolith without metal-ion. The affinity column has shown efficient capture of high abundant proteins such as IgG, transferrin and albumin from human plasma.

Development of a metal/chelate polyhydroxyethylmethacrylate monolith capillary for selective depletion of immunoglobulin G from human plasma for proteomics.

In this study, we report the development of a new poly HEMA (HEMA-co-DEGDA-co-DATD) monolith capillary functionalized with "IDA-Cu (II) complex". Of the two tested crosslinkers (methylene bisacrylamide (MBAAm) and diethylene glycol diacrylate (DEGDA)), presence of DEGDA has enhanced the monolith rigidity. Structural assembly of these monoliths are organized with highly interconnected large globule like structures and dominated by macropore region. Iminodiacetic acid (IDA) immobilization was performed using two chemical approaches (i. aldehyde - secondary amine reaction and ii. epoxy - sec. amine reaction). FT-IR analysis confirmed successful IDA immobilization in both cases. For the first time, a reaction of sec. amine ligand with aldehyde functional material was successfully reported. Overall, the Cu (II)-IDA monolith capillary showed good permeability (3.05×10 -13m2), high IgG adsorption capacity and reusablilty even after 5 consecutive adsorption-desorption cycles. The amount of protein (IgG/HSA) adsorbed on Cu (II)-IDA monolith prepared via the two chemistries is almost similar. Using this affinity monolith capillary, we selectively depleted ∼95% of IgG from human plasma (dilution of 1:16).

Preparation and characterization of a Cu (II)-IDA poly HEMA monolith syringe for proteomic applications.

Herein, we report the preparation of a metal-chelate immobilized hydrophilic poly hydroxyethylmethacrylate-based monolith of 100 µL volume in a plastic syringe. The monolith is elastic in nature, contains well interconnected pores with a permeability (k) of 1.3 × 10-12  m2 . Immobilization of iminodiacetic acid (IDA) is performed via schiff base reaction. Adsortion of IgG on this copper-IDA monolith is of langmuir isotherm with a maximum adsorption capacity of ∼25 mg IgG per g monolith. IgG adsorption capacity of this affinity monolith remained unaffected with increase of flow rate. This proposed metal-chelate monolith in syringe format has the potential for application in proteomics.