Application of molecularly imprinted polymers in food sample analysis – a perspective

Since the introduction of the molecularly imprinting technology (MIT) in 1970s, it becomes an emerging technology with the potential for wide-ranging applications in food manufacturing, processing, analysis and quality control. It has been successfully applied in food microbiology, removal of undesirable components from food matrices, detection of hazardous residues or pollutants and sensors. Molecularly imprinted solid-phase extraction (MISPE) is the most common application so far. The review describes the methods of making the molecularly imprinted polymer systems, the application of the technology in food safety issues and the remaining challenges

Development of Molecularly Imprinted Polymers for Detection of Malachite Green in Cultured Tilapia (Oreochromis Mossambicus) and Catfish (Clarias Batrachus)

Malachite green (MG) is a synthetic dye used for dying leather, silk, cotton and paper. It has been used illegally to treat fungal infection on fish and it persists in fish tissue for relatively long time. Research indicates that MG is carcinogenic. Molecularly imprinted polymers (MIPs) offer an attractive alternative to the conventional detection methods, for example high performance liquid chromatography and liquid chromatography-ion trap mass spectrometry, which are time-consuming and tedious. Molecularly imprinting technology (MIT) is a technique where the polymers are synthesized in the presence of target molecule which acts as the template. The MIPs can specifically bind the target molecule in preference to other closely related molecules. Therefore, the aim of this study was to develop the MIPs targeting MG in order to detect its presence in seafood. A series of MIPs were prepared using MG as the template molecules by non-covalent bulk polymerization. MG, methacrylic acid, ethylene glycol dimethacrylate, azobisisobutyronitrile and acetonitrile were used as template, functional monomer,crosslinker, initiator and porogen respectively. Their physical properties have been characterized using scanning electron microscope. The rebinding capability and selectivity have been evaluated. In the rebinding analysis, the MG adsorption amount increased with the increasing of MG concentration, where the MIPs bound more MG comparing with the control non-imprinted polymers (NIPs). Scatchard plot’s analysis revealed that there was one class of binding sites populated in the imprinted polymers with maximum adsorption capacity of MG of 0.491-0.906 μmol/g and dissociation constants were estimated to be 9.091-20.000 μmol/L. The selectivity test showed that the MIPs could specifically bind the MG comparing with methyl violet,which is structurally similar to the MG. The imprinting factor was in the range of 1.59-1.88. The MIPs have showed ability to rebind MG in walking catfish (Clarias batrachus) and tilapia (Oreochromis mossambicus) for the real sample analysis. MIPs that were synthesized with the presence of MG showed desired results, where their rebinding ability and specificity were better compared with the control NIPs. The data and method will serve as crucial knowledge for the development of MIP biosensor for MG detection in the future study

Silencing BRE Expression in Human Umbilical Cord Perivascular (HUCPV) Progenitor Cells Accelerates Osteogenic and Chondrogenic Differentiation

<div><p>BRE is a multifunctional adapter protein involved in DNA repair, cell survival and stress response. To date, most studies of this protein have been focused in the tumor model. The role of BRE in stem cell biology has never been investigated. Therefore, we have used HUCPV progenitor cells to elucidate the function of BRE. HUCPV cells are multipotent fetal progenitor cells which possess the ability to differentiate into a multitude of mesenchymal cell lineages when chemically induced and can be more easily amplified in culture. In this study, we have established that BRE expression was normally expressed in HUCPV cells but become down-regulated when the cells were induced to differentiate. In addition, silencing <i>BRE</i> expression, using <i>BRE</i>-siRNAs, in HUCPV cells could accelerate induced chondrogenic and osteogenic differentiation. Hence, we postulated that BRE played an important role in maintaining the stemness of HUCPV cells. We used microarray analysis to examine the transcriptome of <i>BRE</i>-silenced cells. <i>BRE</i>-silencing negatively regulated <i>OCT4</i>, <i>FGF5</i> and <i>FOXO1A</i>. <i>BRE</i>-silencing also altered the expression of epigenetic genes and components of the TGF-β/BMP and FGF signaling pathways which are crucially involved in maintaining stem cell self-renewal. Comparative proteomic profiling also revealed that <i>BRE</i>-silencing resulted in decreased expressions of actin-binding proteins. In sum, we propose that BRE acts like an adaptor protein that promotes stemness and at the same time inhibits the differentiation of HUCPV cells.</p></div

HUCPV cells differentiated into osteoblasts and chondrocytes, 3 and 4 weeks after induction, respectively.

<p>(A) Alizarin red S staining and immunofluorescence staining with pro-collagen type-I antibody were used to demonstrate osteogenic differentiation by HUCPV cells maintained in osteogenic (Ost) medium and control (Ctl) media for 3 weeks. (B) Alcian blue staining and immunofluorescence staining with SOX9 antibody were used to demonstrate chondrogenic differentiation in HUCPV cells cultured in chondrogenic (Chon) and control (Ctl) media for 4 weeks. The nuclei were counterstained with DAPI. N = 3 independent experiments.</p

Extraction and purification of HUCPV cells.

<p>(A) Representative picture of a human umbilical cord showing the umbilical vein (represented by solid circle) and umbilical arteries (represented by dashed circle). (B) Prior to treatment with collagenase, the umbilical blood vessel was ligated at both ends. (C) The primary HUCPV cells were isolated by collagenase digestion of the perivascular region of the ligated blood vessel.</p

Comparative proteomics analysis of differentially expressed proteins in BRE silenced HUCPV cells.

<p>A representative silver stained 2-DE gel of total protein extracted from HUCPV cells that had been transfected with <i>BRE</i>-siRNA. When compared with control 2-DE gels, the ESI-MS/MS analysis identified proteins that were up- and down-regulated as a result of silencing <i>BRE</i>. Cytoskeletal binding proteins were especially affected. N = 3 independent experiments.</p

BRE expressions during HUCPV cell differentiation.

<p>(A) RT-qPCR revealed that as HUCPV cells were induced to differentiate in osteogenic (Ost) and chondrogenic (chon) medium, <i>BRE</i> expression was down-regulated. <i>BRE</i> expression was normalized against <i>GAPDH</i>. The statistical difference of P values were determined by t-test; *p<0.05, **p<0.01 and *p<.05 were considered significantly different. (B) Immunofluorescence microscopy confirmed that BRE expression (red) was suppressed as the HUCPV cells were induced to form osteoblasts and chondroblasts. Control (Ctl) medium. Osteogenic (Ost) and chondrogenic (Chon) inducing medium. The nuclei were counterstained with DAPI. N = 3 independent experiments.</p

BRE expressions in <i>BRE</i>-silenced HUCPV cells.

<p>(A) RT-qPCR and (B) immunofluorescence microscopy showing <i>BRE</i>-siRNAs could silence BRE expression in HUCPV cells. Our control <i>Ctl-siRNAs</i> did not affect BRE expression. For RT-qPCR, <i>BRE</i> expression was normalized to housekeeping gene <i>GAPDH</i>. The statistical difference of P values were determined by t-test and **p<0.01 were considered significantly different. The nuclei were counterstained with DAPI. N = 3 independent experiments.</p

BRE and OCT4 expression in ESCs.

<p>(A–C) In the presence of LIF, BRE and OCT4 were strongly co-expressed in undifferentiated ESCs (yellow dotted outlines). (D) LIF was withdrawn from the culture for 24 hours to allow the ESCs to differentiate. This resulted in a reduction of BRE and OCT4 expression in the ESCs (white dotted outlines). N = 3 independent experiments.</p