Application of nanocomposite polymer hydrogels for ultra-sensitive fluorescence detection of proteins in gel electrophoresis

Accepted Manuscript Application of Nanocomposite Polymer Hydrogels for Ultra-Sensitive Fluorescence Detection of Proteins in Gel Electrophoresis Mohammad Zarei PII:

S0165-9936(17)30047-X

DOI:

10.1016/j.trac.2017.05.003

Reference:

TRAC 14922

To appear in:

Trends in Analytical Chemistry

Received Date: 9 February 2017 Revised Date:

31 March 2017

Accepted Date: 15 May 2017

Please cite this article as: M. Zarei, Application of Nanocomposite Polymer Hydrogels for Ultra-Sensitive Fluorescence Detection of Proteins in Gel Electrophoresis, Trends in Analytical Chemistry (2017), doi: 10.1016/j.trac.2017.05.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Application of Nanocomposite Polymer Hydrogels for Ultra-Sensitive

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Mohammad Zarei

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Fluorescence Detection of Proteins in Gel Electrophoresis

Department of Chemical and Civil Engineering,

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University of Kurdistan, Sanandaj, P.O. Box 66177, Iran

■ Author information *

Corresponding Author: Dr. Mohammad Zarei, [email protected]

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Abstract:

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Polymer nanocomposites (PNCs) are a special class of materials which nanoparticles (NPs) dispersed in a polymer matrix resulting in novel materials having unique physical and chemical properties. Successful combination of different properties of the NPs with the existing properties

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of polymers matrix became the goal of scientists. Gel electrophoresis is a gold standard analysis tool used as a routine and important separation technique for proteomics and genomics

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characterization of proteins and DNA samples. Various strategies were proposed for improving the parent method performance including incorporation of nanomaterials. This review focuses on the latest applications and achievements of using PNCs as separation medium in gel electrophoresis. For each type of nanomaterials, we describe different examples. Also, we

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propose strategies for improving the efficiency and influence of NPs in host polymer hydrogel matrix. Further, we discuss potential directions and issues worth exploring for application of

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novel PNCs in gel electrophoresis.

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Keywords: Gel electrophoresis; Polymer nanocomposites; Proteomics; Hydrogel; Nanoparticle; Detection

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1. Introduction With the approach of the post-genome era and integrity of recombinant DNA technology, there has been a resurgence in the application of gel electrophoresis for identification and

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characterization of various genome and proteome products [1]. Slab gel electrophoresis has become the principle tool in analytical chemistry, genetics, proteomics, and molecular biology. Within the last 30 years, various polymer hydrogels and sieving materials have been developed

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to improve the performance of methods. Implemented in slab gels, capillaries, and microfluidic systems hydrogels are cornerstone of proteomics and genomics applications [2]. Polymeric

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separation hydrogels are used to obtain the high separation efficiency in slab gels, capillaries, and microdevice channels. Crosslinked and non-crosslinked polymer gels have been successfully used in slab gel electrophoresis, capillary electrophoresis (CE), and lab-on-a-chip systems. Polyacrylamide gel electrophoresis (PAGE) can be used to analyse the size, amount, purity,

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and isoelectric point of polypeptides and proteins. In order to determine protein molecular size and mass, proteins are treated with the surfactant sodium dodecyl sulphate (SDS) which making slab gel SDS-PAGE an important protein separation tool [3]. Polyacrylamide (PAM) gel is also

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an anti-convective separation medium which produces sharp separated protein patterns with minimal convective dispersion. However, slab-gel electrophoresis suffers from Joule heating,

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diffusion, high sample consumption, poor reproducibility, and time-consuming post-separation analysis and staining [4]. Capillary gel electrophoresis (CGE) was introduced to overcome the limitations of slab-gel electrophoresis [5]. Narrow bore fused-silica capillaries filled with crosslinked gels or non-crosslinked linear polymer solutions lead to ultrahigh resolving power and enhanced performance for separation of complex macromolecules. CGE separates complex mixtures in just minutes with excellent reproducibility, generating a large amount of data. Also,

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CGE systems are capable of rigorous qualitative and quantitative analysis of the separation profiles [6]. Although, at the expense of cost, and the loss of high throughput capability. Separation slab gels are multi-lane, multi-dimensional systems which operate the simultaneous

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analysis of proteins and DNAs at once in separation gels which these approaches are not easy to perform by CE systems. Also, for further and subsequent analysis it is important to fractioncollect the protein and DNA samples, and it is more efficient to obtain an adequate amount of

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proteins or DNAs from slab gel electrophoresis than CE [7]. Therefore, improving the slab gel electrophoresis as a cheap, easy, and high throughput technique is still interesting.

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Various separation sieving matrices have been applied in electrophoresis. Beside the homopolymers, various modified and grafted polymer, tri-block copolymers, nanoporous medium, and interpenetrating networks will be studied further [8]. Stimuli-responsive polymers (block copolymer gels), thermo-responsive pseudogels, and PNCs are emerging materials for

stimuli-response [9].

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separation media with versatile and adjustable properties including pore size, hydrophilicity, and

Nanocomposite polymer hydrogels are interesting materials used for electrophoretic

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separation of biomolecules. Carbon nanotubes (CNTs) were used for improving the efficiency of separation in slab gel electrophoresis and improved the sensitivity and efficiency of separation

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[10, 11]. There is a disadvantage about using CNT because of high thermal conductivity (3500 W m-1 K-1) [12] as well as high electrical conductivity (106-107 S m-1) [13]. Thus, due to high electrical conductivity, CNTs can migrate through the gel matrix during the separation process when voltage is applied which leads to depletion-accumulation of CNTs in different parts of the gel. Another limitation is the black color of CNTs, thus transparent polymer nanowires [14] and semiconductor nanomaterials could replace the CNTs in electrophoresis.

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This review focuses on application of PNCs in gel electrophoresis for improving the fluorescence detection of proteins. Also, aims to provide a basic information about the incorporation of different types of nanomaterials in gel electrophoresis systems. Instead of

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considering historical rarely used separation media, we emphasize on recent novel approaches and nanomaterials. We summarize PNCs hydrogels properties, common or particularly

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noteworthy separation conditions, and interesting applications using these materials.

2. Opportunities of Using Nanomaterials

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NPs are materials have at least one dimension less than 100 nm in size. Various NPs synthesis methods developed and NPs with unique characteristics are being utilized in medical, chemical, and biological applications. Generally NPs classified into carbonaceous nanomaterials [15], metal oxide NPs [16], metallic NPs [17], semiconductor and carbon-based dots [18], polymer

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NPs [19], and inorganic NPs [20] which have been used in separation science [21-23]. Application of nanomaterials in gas chromatography, liquid chromatography, CE, capillary electrochromatography (CEC), and microchip electrophoresis (MCE) have been extensively

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studied [24-26]. Successful application of NPs in separation science encourage the researchers to expand the application to other separation techniques to solve the problems such as Joule

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heating, dispersion, and diffusion. Physicochemical characteristics, tunable properties, controlled chemistries, and ease of modifications are additional advantages of using NPs in electrophoretic separations. Also, NPs can be incorporated, grafted, and anchored with selected polymers and gels for improving the efficiency of protein separations [27]. NPs possess high surface-to-volume ratio which enhances the thermal conductivity and mass transfer and increases the separation efficiency [28]. NPs can be added to the running buffer, and

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polymer solution inside capillary columns which used in CE, CEC, and MCEC techniques [2933]. The NPs can stabilize and decrease the electroosmotic flow (EOF), exhibit reversed-phase behavior, and can be added to polymer solution to reduce the aggregation. Monolith columns

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incorporated with nano-sized metal organic frameworks (MOFs) and hybrid nanomaterials have been successfully used for separations [34]. Also, zeolitic imidazolate framework (ZIF-8)-

derived nanoporous carbons are used in CEC capillary columns [35]. The incorporation of the

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ZIF-8-derived carbons into the organic polymer monoliths led to an increase in the retention of all the analytes compared to the parent monolith and the hybrid monolithic columns exhibited

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satisfactory run-to-run and batch-to-batch reproducibility. Due to small size, various shapes, high surface-to-volume ratios, and favorable surface chemistry (Fig. 1), NPs are extensively being used in separation techniques to improve the features of parent techniques. Organic–inorganic hybrid nanomaterials showing promising application potentials in

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chemistry, medicine, and materials science [36]. As a result of organic–inorganic combination, hybrid nanomaterials assemble numerous extraordinary characteristics such as stability, enhanced optical properties, biocompatibility, and other physical and chemical properties [37].

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Semiconductor quantum dots (SQDs) as ideal fluorescent probe with unique properties and high fluorescence quantum yields were employed for fluorescence imaging for various applications.

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However, the toxicity of SQDs raises questions about biohazard and accumulation issues [38]. Hence, the design and development of novel hybrid nanomaterials with properties such as high fluorescence quantum yield, strong photo stability and excellent biocompatibility are of great significance [39]. The fabrication of a multicomponent hybrids were emerged as a new approach for the enhanced florescent detection.

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Figure 1. Ligand exchange and functional groups of gold NPs and quantum clusters. Reprinted from [40] with permission.

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The potential to be functionalized by a wide range of different chemistries and functional groups such as amino, phosphate, sulfhydryl, thiol, and carboxyl increased the range of NPs

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application in separation science (Fig. 1). Tightly-packed, perfectly-aligned, and monodispersed NPs introduced a novel nanomaterials for improving the separation process [41]. The ideal NPs must be electrically charged to avoid the coelution with the EOF, give small mass transfer resistance to reduce the band broadening, show less interference with detection, and be porous and small to provide a high surface area to improve sample capacity [42]. These considerations and characteristics are crucial for NPs to improve the separation efficiency [42].

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3. Nanocomposites Polymer Hydrogels Polymer hydrogels are widely used to produce biomolecular separations in electrophoresis, where gel morphology and charge effects combine to produce an efficient separation (see Table

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1). A typical polymer composite is a combination of a polymer matrix and a filler. Because

compounding is a technique that can ameliorate the drawbacks of conventional polymers, it has been studied over a long period and its practical applications are well known. A filler, typically

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micron-sized or nano-sized, is incorporated into composite materials to improve their properties. The polymer matrix and the fillers are bonded to each other by weak intermolecular forces, and

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chemical bonding is rarely involved. If the reinforcing material in the composite could be dispersed on a molecular scale (nanometer level) and interacted with the matrix by chemical bonding, then significant improvements in the mechanical properties of the material or unexpected new properties might be realized.

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Incorporation of nanomaterials into the host matrix introduces an interesting and unique characteristics to host gel matrix. Also, some host matrix properties improved or elevated by NPs incorporation. Also, presence of NPs can alter to the gel morphology and physical

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characteristics. In Fig. 2, various scales relevant to NPs incorporation in polymer hydrogels are depicted. At the macroscopic scale, the opacity and cross-link heterogeneity nanocomposite

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hydrogel performance can differ from pure hydrogel. At the media scale, the nanocomposite can act to improve the gel strength and elongation. At the micro channel scale, NPs can interact with the analyte by forming channel walls whose localized charge and geometry will be different from those of the pure hydrogel. At the nanoscale, the NPs can force transport of the analyte through interparticle channels. Further, the incorporation of NPs can alter the gel cross-link density, electrical, and thermal conductivity of host matrix [43]. Given the extensive variety of NPs now

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accessible (clays, CNTs, graphene, graphitic carbon nitride, silica, titania, zirconia, and various oxides, etc.), the potential combinations of polymers and NPs, and thus the tailor ability of the property suite, is essentially endless. Despite the widespread application of nanomaterials in

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separation science, very little research has been devoted to the application of nanomaterials in slab gel electrophoresis and still there is need to conduct more researches in this field. In this section we discuss the reported nanomaterials for improving the separation and detection of slab

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gel electrophoresis.

Figure 2. NPs incorporation can affect the gel thermal conductivity, strength, opacity, and matrix heterogeneities. NPs change the cross-link density, pore structure, and electrical double layers at microchannel scales and nanoscales. Reprinted from [43] with permission.

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Table 1. Molecular sieving polymer, hydrogels, and PNCs for electrophoresis. Reference

Standard proteins or mixtures, A and B chains of insulin, protein samples (20– 116 kDa), serum lipoproteins

[5, 44-48]

Method

Comment

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Inchannel/capillary PAM

Analyte

SDS-PAGE CE, MCE, 2-DE, CE

Rapid separation, high efficiency, linear and nonlinear decreasing pore-size gradients in cross-linked PAM gels, no need for controlled mixing and delivery of polymer precursor solutions, protein sizing in ultrashort separation lengths, generic, inexpensive, versatile, good resolution

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Separation matrix*

Standard proteins or mixtures

[49-52]

CE, CZE, MCE, capillary array electrophoresis bioprocessor

Most widely used matrix, viscous, high separation resolution, optically transparent, strong UV absorbance, fluorescence background, toxic

PDMA

Standard proteins

[53, 54]

Capillary sodium dodecyl sulfate-gel electrophoresis, microfabricated capillary array electrophoresis microplates

Low viscosity, lower resolution than linear PAM, hydrophobic, high surface coating capacity

PVP

CA isoforms

[55]

CE

Low viscosity, lower resolution than linear PAM, high surface coating capacity, hydrophobic

Beckman SDS gel

Apolipoproteins, (rMAbs), standard proteins, protein from E. coli cell, protein biotoxins

[48, 56-63]

CGE, capillary sodium dodecyl sulfate gel electrophoresis, 2-D microchip electrophoresis

Improved performance and throughput, used for antibody development including bioprocess optimization, antibody characterization, release, and formulation stability assessment, no interference from sample buffer matrix, linear, accurate, and precise in the range of protein concentration

Dextran

Proteins standard, BSA

[64, 65]

MCE, microchip SDSgel electrophoresis

Low viscosity, low UV absorbance, high separation resolution, weak surface coating capacity, heat required to improve separation

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Linear PAM

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Standard proteins, glycoproteins and deN-glycosylated human serum glycoproteins, proteins from soybean cultivars

[66-69]

Microchip CGE, CGEon-a-chip, SDS gel CE

Shorter analysis time and an increased robustness and ease of use, extended protein separation range, suitable for monitoring the de-N-glycosylation progress

BSA gels crosslinked with glutaraldehyde

Tryptophan enantiomers

[70]

CE

Bio-Rad CE-SDS run buffer

Polyethylene glycolylated interferon (PEG-IFN)

[71]

CE

Does not require coated capillaries for SDS-CGE. The SDS–CGE method showed high separation capacity by differentiating PEG-IFN conjugates with small differences in molecular size, and it was useful for checking the purity of each mono-PEG-IFN.

Slightly crosslinked PAM

E. coli AcrA protein, proteins in crude cell extract

[72, 73]

Sodium dodecyl sulfatecapillary PAM Gel Electrophoresis,

Increased separation efficiency, reduced separation time, and automated operation. Compared to linear polyarylamide, poly (ethylene oxide) and hydroxypropyl cellulose, the new matrix permitted the highest resolutions with comparable or increased separation speed.

PEG/dextran

Proteins in MCF-7 breast cancer cell, proteins in Barrett’s Esophagus Tissue homogenate

[74, 75]

CE, 2-D CE

Rapid and reproducible separations of Barrett's esophagus tissue homogenates. The first capillary employs capillary sieving electrophoresis using a replaceable sieving matrix. Fractions are successively transferred to a second capillary where they undergo additional separation by micellar electrokinetic capillary chromatography

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Agilent 2100 kit

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Useful for affinity separation in electrophoresis. BSA gel used as the chiral selector for the separation of tryptophan enantiomers with high efficiency compared to the analogous protein-based LC separation

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Standard proteins, Deinococcus radiodurans protein, native MC3T3-E1 cells

[76-78]

(1-D and 2-D) CE, 2-D CGE, capillary sieving electrophoresis/micellar electrokinetic capillary chromatography

Low viscosity, low UV absorbance, improved separation resolution, weak surface coating capacity, efficient protein separation in analysis times

MC

TI, BSA, amylogluocosidase/ BSA/BSA-antibody complex

[79]

MCE

High surface coating capacity, hydrophobic

HPMC

Lipoproteins HDL, LDL, VLDL,bsa, standard protein

[80, 81]

MCE, isotachophoretic gel electrophoresis

Low viscosity, high surface coating capacity, needs additives to improve separation

PEO-PPO-PEO

Oligonucleotide standard (8–32 base)

[82]

Microchip-based CE

Thermo-responsive character, adjustable pore structure, limited to DNA separation, temperature dependent viscosity-adjustable property

PAM

Standard protein, human sera

[83, 84]

2-D microfluidic electrophoresis, microfluidic protein electrophoresis, freestanding PAM gel (fsPAG) microstructures

Diverse chemical modification, capable of photopatterning, heterogeneity at nanoscale, high efficiency and minimal sample dispersion, The facile fabrication and prototyping of the fsPAGE, enhancement in separation performance

Polymethacrylate

Protein standard, BSA tryptic digest

[85, 86]

Electric field gradient focusing (EFGF), polymer microfluidic chips

Low interaction with DNA or proteins, biocompatible, capable of photopatterning, low gel mechanical strength, difficult to refresh, heterogeneity at nanoscale, increase detection sensitivity and separation performance

PNIPAAm

Protein standard

CGE

Superior thermoresponsive character, more hydrophobic than PAM, protein adsorption, using such a copolymer, one can easily realize high-speed, high-efficiency and reproducible capillary gel electrophoresis for proteins without any instrumental

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Pullulan

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[87]

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rebuilding or investment Bovine serum albumin and lysozyme

[88]

Ultrafiltration, CE

PEO

Proteins in HT29 human colon adenocarcinoma cell extract, Actin, OVA, protein A, BSA, helix pomatia lectin, Concanavalin A, lectin peanut agglutinin

[89, 90]

Capillary sodium dodecyl sulfate (SDS)DALT electrophoresis (SDS-DALT-CE), 2DSDS microcapillary gel electrophoresis (SDS µCGE)

High surface coating capacity, low viscosity, low UV absorbance, highly hydrophobic, nonreactive with fluorogenic labelling reagent, separation comparable with PAGE, weak interaction to DNA or proteins, low gel mechanical strength

Poly-Nhydroxyethylacryl amide

Standard proteins

[91]

MCE

Significantly decrease the time and increase the attainable level of automation and integration of the front-end protein fractionation, use of poly-Nhydroxyethylacrylamide as a dynamic, hydrophilic chip channel coating that can be applied with a rapid and simple protocol for size-based protein separation

HPC

Standard proteins or mixtures Lipoproteins HDL, sdLDL, Lldl, HT29 human colon adenocarcinoma cell extracts

[92, 93]

DHPC-DMPC

DNA fragments

[9]

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Thermo-responsive (viscosity changing) polymer, excellent dynamic surface coating, incompatible with dynamic labelling dye, require higher concentration for separation than hydroxyethylcellulose does, separation of serum small, dense low-density lipoprotein, high separation speed and high reproducibility

CE

Thermo-responsive characteristic is useful for tunable sieving based electrophoretic separations (e.g., of DNA), useful as sieving agent in microscale channels. Advantages of utilizing phospholipid pseudogels for

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Selectivity for the separation of the binary protein mixture could be systematically increased with increasing membrane charge density. Due to their high stability and tunable functionality, the PAES block copolymers have also large potential as membrane material for other applications.

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PAES

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sieving are the ease with which they are introduced into the separation channel and the potential to implement gradient separations Lanthanide chelatelabeled proteins Fluorescence proteins RPE, BPE and GFP, BSA

[94]

SDS-PAGE, CGE

Low viscosity, robust entangled network at low concentration, required purification, gel after electrophoresis can be dried for further analysis

Ceria/PAM

Standard protein, E.coli protein

[95]

SDS-PAGE

Zirconia/agarose

Standard DNA samples

[96]

Agarose gel electrophoresis

High thermal conductivity, lower Joule heating, faster separation, high efficiency, increased UV background, NP concentration gradient

Tungsten oxide/agarose

Standard DNA samples

[96]

Agarose gel electrophoresis

High thermal conductivity, lower Joule heating, faster separation, increased UV background, NP concentration gradient

CNT/PAM

Serum and liver protein samples, standard proteins

[10, 11]

GCN/PAM

E.coli proteins

[97]

Au/PAM

Au/P(NIPAM-co-

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HEC

Lower viscosity than PAM, high thermal conductivity, lower Joule heating, faster separation, high efficiency

Improved separation, could be applied for the detection of sera from patients with liver diseases, improve resolution in the detection of human serum with native PAM gel electrophoresis system

SDS-PAGE

High thermal conductivity, lower Joule heating, faster separation, high efficiency, biocompatible, improved catalytic behavior

BSA, standard proteins [98, 99]

1-D and 2-D PAGE

Improved sensitivity, high efficiency and resolution, sensitivity of 7–14 times higher than that of traditional staining detection methods

Standard proteins

MCE

Optomechanically responsive nanocomposite

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SDS-PAGE

[100]

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hydrogels is achieved using strongly absorbing Au NPs or nanoshells embedded within a thermally responsive polymer. Valves formed from composites with different NPs could be independently controlled by changing the illumination wavelength

DNA samples

[101]

Nanoparticle-filled CE (NFCE)

Separation of high molecular weight DNA markers (8.27−48.5 kbp) with plate numbers greater than 106 suggests, promising for the analysis of long-stranded DNA molecules such as chromosomes, simple and affordable when compared to those that use microand nanofabricated devices for separating long DNA molecules

Si SQD/PAM

Human serum proteins

[102]

PAGE

High resolution, sensitive and dependable method for direct detection of human serum proteins, and has enormous potential in clinical diagnosis

CdTe SQD/PAM

Human serum proteins

[103]

PAGE

Stable signal, high resolution, and high sensitivity for the simultaneous detection of human serum proteins on gels after PAGE separation

Ag/PAM

Human serum proteins

[104]

Au/PDADMAC

Aminophenols

[105]

Au/polyimide

Human serum proteins

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Au/PEO

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AM)

[106]

PAGE

Fast, nontoxic and sensitive, shows great analytical potential in proteome research and in biochemistry

MCE

Improve the selectivities and efficiency of the separation. Electrochemical detection and the quantitation of the solutes were not affected by the PDADMAC and the Au NPs

Chemiluminescence immunoassay hyphenated to CE (CEbased CL-IA)

Sensitive chemiluminescence immunoassay by capillary electrophoresis, Au NPs were used as a protein label reagent in the light of its excellent catalytic effect to the CL reaction of luminol and hydrogen peroxide

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Acidic proteins, protein glycoforms and bioactive peptides

[107]

High-resolution separation of proteins and peptides, a substantial improvement of separation efficiency in particle-coated capillaries was observed

CE

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Polystyrene/ polyhydroxyl

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* See list of abbreviations.

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3.1. Metallic PAM Nanocomposites Metallic SQDs and nanoclusters (NCs) are used for improving the separation of protein samples. SQDs are extensively used in biological research because of their unique properties,

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such as size-tunable emission, high quantum yield, tunable photoluminescence (PL), broad

excitation spectra and narrow emission. Due to their electric surface charge, SQDs are well

suited for electrophoretic separations take benefit from the photoluminescent characteristics of

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SQDs by using SQDs as fluorescent labels for detection purposes [108, 109]. Noble metal NCs (NCs; Nanoclusters are ultrafine particles of nanometer dimensions) of sizes comparable to the

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Fermi-wavelength of the conduction electrons show molecular-like characteristics such as luminescence properties. NCs are new class of fluorophores because of the bright fluorescence which NCs emit following their discrete and size-tunable electron transition from the excited states [110]. Compared with the SQDs, which are larger in dimension and typically exhibit

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toxicity, noble metal NCs are attractive because of their photo stability, small size and nontoxicity. Zhang and co-workers applied a fluorescence imaging detection technique for improving the sensitivity of proteins detection in PAGE [98]. The BSA modified gold (Au) NCs

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acted as fluorescent probes for fluorescence imaging (Au NCs ~ several to tens of atoms [98]; Au NPs (3nm) ~ 500 atoms [111]). The Au NC-based fluorescence detection method showed

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lower interference, background, and higher sensitivity compared with traditional techniques. Fig. 3A shows 1-D PAGE gels of a series of human serum protein samples, with different concentrations. Gels were stained by the three detection methods: (a) BSA-stabilized Au NCbased fluorescence imaging, (b) silver staining, and (c) CBB-R250 stain. Result showed the Au NC-based fluorescence imaging (Fig. 3A-a) can even detect at low dilution ratio (lane 8), which

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Figure 3. (A) Comparison of sensitivity: (a) BSA-stabilized Au NC-based fluorescence imaging, (b) silver staining and (c) CBB-R250 stain. The dilution ratios of the serum samples (from left to

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right) are as follows: 1/2(1), 1/5(2), 1/10(3), 1/20(4), 1/40(5), 1/80(6), 1/100(7), 1/200(8), 1/300(9) and 1/400(10). (B) Enhanced fluorescence comparison (a) fluorescence image with oxygen LTP-treated BSA-stabilized Au NCs. (b) A direct BSA-stabilized Au NC-based fluorescence image. The dilution ratios of the serum samples (from left to right) are as follows: 1/2(1), 1/5(2), 1/10(3), 1/20(4), 1/80(5), 1/100(6), 1/200(7), 1/300(8), 1/400(9) and 1/500(10). (C) The detection of human serum proteins after 2-D PAGE by (a) BSA-stabilized Au NC-based fluorescence imaging (b) CBB-R250 stain. Reprinted from [98] with permission.

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was similar to the silver staining (Fig. 3A-b) whereas the protein were hardly detectable at a 1/80 dilution ratio (lane 6) in CBB-R250 staining (Fig. 3A-c, dashed boxes). Low-temperature plasma (LTP) treatment of the Au NCs was applied to enhance sensitivities the fluorescence imaging.

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Proteins can be detected at ratio of 1/200 (lane 7) by Au NC-based fluorescence imaging (Fig. 3B-b) and at ratio of 1/400 (lane 9) in the oxygen LTP enhanced fluorescent image (Fig. 3B-a). Fig. 3C shows 2-D gel electrophoresis patterns for separation of proteins. Two dimensional gel

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electrophoresis is a classical gel electrophoresis technique for the analysis and separation of protein and DNA samples. Compared with 1-D electrophoresis, 2-D can provide much more

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information and analysis on the protein and DNA composition in samples. Fig. 3C shows a performance of Au NC-based imaging (Fig. 3C-a) and traditional CBB-R250 staining detection (Fig. 3C-b). The Au NC-based method revealed protein details (regions 1, 2, 3, and 4) that were not detected by the traditional CBBR-250 staining. Also, proteins with low concentration were

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detected in the Au NC-based patterns. According to the result, the resolution and selectivity were improved by application of BSA-stabilized Au NC-based fluorescence method. Na and co-workers [99] described the use of Au NPs for “turn on” fluorescence imaging of

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in-gel proteins after PAGE. The Au NP-based fluorescence imaging was applied for the detection of human serum proteins after 1-D and 2-D electrophoresis and CBB-R250 staining

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was used for comparison. Comparison of Au NP-based imaging and CBB-R250 staining of human serum after 2-D electrophoresis showed higher signal intensity and resolution was obtained by Au NP-based imaging. Also, influence of NPs size on the optical characteristics was investigated by incubation of Au NPs with serum proteins. Result showed different sizes of Au NPs showed different increased fluorescence signals, and the larger sizes of Au NPs gave lower increased fluorescence intensity. By increase of Au NP size from 10 nm to 65 nm, the

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fluorescence intensity decreased. This significant fluorescence emitted from in-gel proteins incubated by Au NPs could be used for the in-gel detection of proteins. The Au NP-based fluorescence imaging method can be used for direct in-gel imaging of proteins after

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electrophoresis. Due to the lower background, this method offered higher resolution and sensitivity compared with CBB-R250 imaging.

Liu and co-workers used amine-terminated silicon (Si) SQDs for the in-gel detection of

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human serum proteins after PAGE [102]. Human serum proteins analyzed and separated by 1-D and native 2-D electrophoresis were detected by Si SQD-based fluorescent imaging, CBBR250

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and silver staining separately (Fig. 4). According to the results Si SQD-based fluorescent imaging method showed low background and clear bands of human serum proteins. Compared with standard methods, the Si SQD-based method showed higher resolution and provided more information than that of CBB-R250 and silver staining methods. This is probably due to the

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unique fluorescent characteristics of Si SQDs. Also, 2-D PAGE was applied to compare the method with traditional CBB-R250 staining and silver staining. As shown in Fig. 4 (boxed regions), the sensitivity and resolution of Si SQD-based fluorescent imaging is higher than that

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of CBB-R250 staining. Moreover, the protein spots a, b and c (marked with arrows) were detected by Si SQD-based fluorescent imaging (Fig. 4A), but could not be evaluated by CBBR-

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250 staining (Fig. 4B. by using Si SQD-based method, some proteins, such as isoform1 of a-1antitrypsin, complement C3 and hemopexin, which were identified by mass spectrometry (MS), were easily detected by using Si SQDs, but not with CBB-R250 staining.

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Figure 4. Detection of human serum proteins after native 2-D electrophoresis by: A) Si SQDbased fluorescent imaging, B) CBB-R250 staining, C) silver staining. Reprinted from [102] with permission. 21

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Na and co-workers used cadmium telluride (CdTe) SQDs for fluorescence imaging and detection of serum proteins on gels after PAGE separation [103]. The thioglycolic acid (TGA) was used to stabilize the CdTe SQDs. Fig. 5 I-A shows clear fluorescent protein bands by the

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CdTe SQD-based fluorescence imaging technique. Comparison of CBB-R250 staining (Fig. 5 IB) with silver staining (Fig. 5 I-C) and CL imaging (Fig. 5 I-D) shows the CdTe SQD-based fluorescence imaging technique provided higher resolution and more protein information.

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Furthermore, the signal intensity of CdTe SQD-based imaging was higher compared with

SYPRO Ruby staining (Fig. 5 I-E). This is due to the unique fluorescence characteristics of

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SQDs such as high quantum yield and high molar extinction coefficient with low background [112]. Fig. 5 II shows the separation gels serum proteins serially diluted from 1:2 to 1:400 of the original sample which stained by the four detection methods. Fig. 5 II-A shows the protein bands at different protein concentrations were recorded by SQD-based imaging, and the transferrin

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marked with an arrow can still be detected even at lane 9, a 1:300 dilution. In CBB-R250 staining method, the bands are hardly detected in lane 6 (Fig. 5 II-B), and cannot be observed in lane 7 for SYPRO Ruby staining or in line 8 for silver staining (Fig. 5 II-C and D, dashed

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rectangles). According to the results, intensities and resolution of transferrin were ranked as: SQD-based fluorescence imaging > CBBR-250 staining > silver staining > SYPRO Ruby

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staining.

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Figure 5. I) Detection of human serum after 1-D PAGE by: A) CdTe SQD-based fluorescence imaging; B) CBB-R250 staining; C) silver staining; D) CL imaging; E) SYPRO Ruby staining. Human serum was diluted 1:10 in the sample buffer (6.67% glycerin and 0.05% bromophenol blue). Loading volume: 15µL. II) Sensitivity comparison of A) CdTe SQD-based fluorescence imaging, B) CBB-R250 staining, C) SYPRO Ruby staining, and D) silver staining. Dilution ratio of the serum samples (from left to right): 1:2 (1), 1:5 (2), 1:10 (3), 1:20 (4), 1:40 (5), 1:80 (6), 1:100 (7), 1:200 (8), 1:300 (9), and 1:400 (10). Loading volume: 15µL. Reprinted from [103] with permission. 23

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The CdTe SQD-based fluorescence imaging technique showed lower background and offered higher resolution and sensitivity than traditional method of CBB-R250 imaging which can be used for the direct detection of human serum proteins on the gel after PAGE.

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Wang and co-workers described application of Ag NCs in fluorescent imaging of human serum proteins after native PAGE [104]. Ag NCs stabilized with Oligonucleotides and used as fluorescent probes for fluorescent detection of proteins after native PAGE. Oligonucleotide-

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stabilized Ag NCs-based fluorescent imaging was used for detection of low-abundance proteins, such as a-1-antichymotrypsin (ACT) and a-2-glycoprotein 1, zinc without the need of expensive

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antibodies or harsh immunoassay protocols. Human serum proteins were analyzed and separated by 1-D PAGE and then detected by three methods: a) CBB-R250 staining; b) silver staining; c) Ag NCs-based fluorescent imaging. Fig. 6 II shows the clear protein bands which are detected by Ag NCs -based fluorescent imaging. The Ag NCs-based fluorescent imaging method provided

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higher resolution in comparison with traditional CBB-R250 and silver staining. Fig. 6 II shows the comparison of Ag NCs -based fluorescent imaging with CBB-R250 and silver staining for analysis of a serial dilution of serum proteins. Fig. 6 I A-C shows sensitivities increased by using

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Ag NCs-based fluorescent imaging. Transferrin can be detected in lane 8 (1:200 dilution) (Fig. 6 I-C). In CBB- R250 staining, this band is only recorded in lane 5 because of lower sensitivity

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(Fig. 6 I-A). In silver staining, this band is detected in lane 7 (Fig. 6 I-B), but it is not clear which this is caused by higher background. Fig. 6 I A-C shows, the sensitivity of the Ag NCs based fluorescent imaging technique is higher than CBB-R250 and silver staining methods.

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Figure 6. I) Sensitivity comparison of: A) CBB-R250 staining; B) silver staining; C) Ag NCs based fluorescent imaging. Dilution ratio of the serum samples were 1:2 (1), 1:5 (2), 1:10 (3), 1:20 (4), 1:40 (5), 1:80(6), 1:100 (7), 1:200 (8), 1:300 (9), and 1:400 (10); loading volume: 15 µL. Detection of human serum after 2-D PAGE by: D) CBB-R250staining; E) silver staining; F) oligonucleotide-stabilized Ag NCs based fluorescent imaging. Sera were diluted (1:6) in the sample buffer II) Detection of human serum after 1-D PAGE by: A) CBB-R250staining; B) silver staining; C) Ag NCs -based fluorescent imaging. Sera were diluted (1:10) in the sample buffer; loading volume: 15 µL. The concentration of DNA oligonucleotide-stabilized Ag clusters was 180 µm. Reprinted from [104] with permission. 25

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Native 2-D PAGE were performed for comparison of these methods. In-gel proteins were detected by CBB-R250 staining, silver staining and Ag NCs -based fluorescent imaging (Fig. 6 I D-F). Fig. 6 I-D shows CBB-R250 only detect a small amount of protein. With silver staining a

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new range of proteins were detected, but protein spots could hardly be separated (Fig. 6 I-E, boxed regions) because of lower resolution. Fig. 6 I-F shows Ag NCs-based imaging technique provides more protein information in human serum samples and more proteins could be detected

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by fluorescence when was used. Also, some test were carried out to obtain the optimal results. The signal intensity changed when the pH was increased from 2.0 to 10.0. The optimal pH was

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chosen at 3.4 with comparison of images at different pH values (2.0, 4.8, 6.2, 8.0, and 10.0).

3.2. Carbonaceous PAM Nanocomposites

In recent years, a large number of allotropic carbonaceous nanomaterials were described in

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the literature, including CNTs, fullerenes, nanodiamonds, graphene, carbon nanofibers, nanohornes, cones, graphene quantum dots (GQDs), carbon quantum dots (CQDs), and carbon nanodots (CNDs) [30, 113]. Generally, carbonaceous nanomaterials showed high mechanical

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strength, high elasticity, high electrical conductivity, and high thermal conductivity [114].

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Whereas, strong van der Waals forces hamper the dispersion and the solubility of carbon-based nanomaterials. Huang and co-workers the functionalized CNT with bovine serum albumin (BSA) proteins [115]. Also, noncovalent functionalization by surfactants was reported [116]. The surface chemistry of the CNT was changed from hydrophobic to hydrophilic. The hydrophilic CNT-surfactant conjugate interacts with the hydrophilic surface of the reductase enzyme in water. These results demonstrate that CNT-surfactant conjugates can interact with proteins in

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aqueous environments, which provides a possibility to separate proteins using PAM gel incorporated with CNT-Triton conjugate. CNTs were used for improving the efficiency of slab gel electrophoresis [10, 11]. Huang and

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co-workers [10] synthesized a PAM nanocomposite gel functionalized by CNT-Triton X100 conjugates. According to the results CNT-PAM nanocomposite gel improved the separation of apolipoprotein A-I and complement C3 proteins due to interaction of CNTs with the proteins.

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Generally, two-dimensional (2-D) electrophoresis or chromatographic methods such as LCMS/MS used for separation of apolipoprotein A-I and complement C3 [117]. CNTs–Triton

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conjugate acts as a selective modifier to change the migration speed of the proteins, the relative selectivity of different proteins being achieved. Huang and co-workers, also included the nanoAl2O3 and nano-TiO2 into the separation gel. By inclusion of these NPs the observed mobility of bands changed, but no significant improvement of relative selectivities observed. The separation

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of apolipoprotein A-I and complement C3 was performed by native PAGE using CNT-PAM gel, which according to literature had only been separated so far by using two dimensional electrophoresis, or other complicated methods. Different concentrations of CNTs (0.100, 0.300

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and 0.400×10−3 gmL−1) were incorporated into PAM gel [10]. No obvious changes were found at concentration of 0.100×10−3 gmL−1. The relative selectivity improved with increasing

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concentrations of CNTs. However, when the concentration of the CNTs is higher than 0.500×10−3 gm−1, the CNT will deposit on the bottom of the gel. At concentration 0.400×10−3 gmL−1, deposition was sometimes observed but no significant deposition was observed when the concentration of the CNTs was below 0.300×10−3 gmL−1. Guo and co-workers [11] described the use of CNTs for improving the efficiency of serum proteins separation by gel electrophoresis. Triton X100 treated single-walled CNT (SWNTs) and

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carboxylic multi-walled carbon nanotube (c-MWNTs) were loaded into different parts of the gel. According to the results, SWNTs selectively adsorbed the proteins and reduced the background of the electropherogram, resulting in sharper and cleaner proteins bands. The migration rate of

addition of c-MWNTs to the specific region of the gel.

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proteins was changed by addition of c-MWNTs and the separation of proteins was improved by

By inclusion of c-MWNTs (0.30 × 10-3 g mL-1) to PAM gel in the specific region, four bands

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were detected, whereas only two bands were detected in unmodified PAM gel. Also, by

incorporation of c-MWNTs into the specific region of the resolving gel with an appropriate

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concentration the resolution and efficiency of separation were improved. The CNTs enhanced the proteins isolation in specific regions of the gel which could not be achieved by the normal PAM gels. The efficient CNT concentration for separation was chosen between 0.2-0.5 × 10-3 g mL-1. No obvious changes in efficiency were obtained at concentrations lower or higher than

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optimum value. Therefore, the c-MWNTs concentration should be carefully chosen during the procedure to carry out an efficient separation.

Na and co-workers [118] used the fluorescent carbon nanodots (CNDs) for fluorescence

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imaging of human serum proteins and E. coli proteins after PAGE. Fig. 7 I shows the CNDs have higher sensitivity on SDS gels than on native gels which might be obtained from the good

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stability of SDS for CNDs [119, 120], which better sustained the fluorescence signals of the CNDs for protein imaging. Fig. 7 I shows the sensitivity of the CNDs method is higher than the CBB-R250 method, which is comparable with silver staining. According to the obtained results, the sensitivity of CNDs imaging method was 8 times higher than the traditional CBB-R250 method. Furthermore, the proteins detection on gels by CNDs method only required 3 h, and the signal of the proteins can be sustained for even longer than 7 days. Quick detection of proteins

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could be achieved when the CNDs were applied for staining under acidic conditions, which is

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environmentally friendly due to the absence of heavy metals in the CNDs preparation.

Figure 7. I) Comparison of sensitivity after (A) SDS-PAGE and (B) native PAGE: (a) CNDs fluorescence imaging, (b) CBB-R250 staining, and (c) silver staining. II) Serum separation by

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native 2-DE. (A) CNDs fluorescence imaging, (B) CBB-R250 staining, and (C) silver staining.

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Reprinted from [118] with permission.

Fig. 7 II shows the 2-D electrophoresis images obtained by different detection methods. The results showed using CNDs fluorescence imaging more proteins in samples could be detected (the rectangular regions shown in Fig. 7 II A) than using CBB-R250 staining (Fig. 7 II B). After silver staining (Fig. 7 II C), some protein spots could hardly be detected because of their low resolution. This CNDs fluorescence imaging presented a simple, fast, and sensitive method for proteome researches. 29

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Parthasarathy and co-workers described the using CNT composites of PAM for separation of proteins by SDS-PAGE [121]. A comparison of the separation patterns by SWCNT/PAM and MWCNT/PAM composite gels showed that the MWCNT/PAM composite gels are more

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efficient in terms of resolution and efficiency. Nevertheless, both types of SWCNT/PAM and MWCNT/PAM composite gels presented higher separation efficiency compared with the pure PAM gel.

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The E. coli proteins were separated in pure PAM and in PAM/GCN gels [97]. By

incorporation of GCN nanosheets into the PAM gel, the resolution and detection limits of protein

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separation increased. Reduction in peak widths and also staining process in the presence of gC3N4 nanosheets cause increase in signal intensity. Also, different concentrations of GCN were incorporated into PAM gel (0.01, 0.03, and 0.04 % (m/v)). No obvious improvement in efficiency was found for GCN concentration of 0.01 % (m/v). The optimum separation efficiency

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was observed for 0.04 % (m/v) of GCN nanosheets [97].

3.3. Metal oxide PAM Nanocomposites

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Metal oxides play a very important role in many areas of chemistry, physics and materials science. The metal elements are able to form a large diversity of oxide compounds. These can

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adopt a vast number of structural geometries with an electronic structure that can exhibit metallic, semiconductor or insulator character. Oxide NPs can exhibit unique physical and chemical properties due to their limited size and a high density of corner or edge surface sites [122].

Semiconductor NPs with high thermal conductivity were used for improving the separation efficiency of electrophoresis [95-97, 123]. The CeO2 NPs have higher thermal conductivity compared with that of the PAM gel but low electrical conductivity (0.044 S m-1) [124]. The 30

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thermal conductivity of bulk CeO2 (19 W m-1 K-1) [125] is significantly higher than that of PAM gel (0.56 W m-1 K-1) [126]. The thermal conductivity of the metal oxide NPs increases with

by NP modified gel at high voltages and temperatures.

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temperature as well as the volume fraction of NPs [127]. This leads to reduction of Joule heating

Standard proteins were separated using both PAM gel and PAM/CeO2 gels at threshold voltage (Vt) for PAM (150 V) [95]. Then, separation was performed at the same conditions at Vt

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(200 V) for PAM/CeO2 gels. By separation at Vt of 150 V and 200 V for PAM and PAM/CeO2 gels, the separation time decreased from maximum 100 minutes to 60 minutes, respectively.

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Also, the separation time of standard proteins was decreased from 33 min in PAM gel at 150 V to 12 min in PAM/CeO2 gel at 200 V. According to the results, theoretical plate number increased in PAM/CeO2 gel compared to those of PAM gel. The resolution improved by increased efficiency and higher applied voltages (Rs ~ N1/2 ~ V1/2) [128]. Overall resolution

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between bands increased in PAM/CeO2 gel compared with pure gel [95]. Also, result suggested that PAM/CeO2 gel image has a higher contrast compared with PAM gel and detection of protein

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bands improved by incorporation of CeO2 NPs.

4. Challenges and Limitations

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Electrical conductivity. Observed deposition and movement of CNTs on the bottom of gel was due to influence of electrical field on electrically conductive CNTs and did not depend on nanomaterial concentration. CNTs have high thermal conductivity [12] as well as high electrical conductivity [13]. Thus, due to this significant electrical conductivity, CNTs can migrate through the gel matrix during the separation process when voltage is applied which leads to depletionaccumulation of CNTs in different parts of the gel. Therefore, choosing the appropriate

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nanomaterials for application in electrophoretic separation can be a very challenging task. Higher thermal conductivity and lower electrical conductivity are two important features which are suitable for improving the efficiency of gel electrophoresis. Although semiconductor

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nanomaterials and SQDs are interesting candidates but, thermal conductivity of semiconductor metal oxide nanomaterials is lower than carbon-based nanomaterials [12, 129].

Distribution. Even and homogeneous distribution of NPs through the gel is essential for

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improving the separation performance. Uneven distribution of NPs through the gel matrix cause the appearance of parabolic temperature profile in gel which temperature in the center of gel is

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higher than edges. Also, viscosity of gel changes by the parabolic temperature profile and there will be an uneven viscosity in different parts of the gel which disturbs the protein migration and enhanced the diffusion process. The uniform distribution of NPs in gel causes the uniform dissipation of heat. Thus, it lowers the uneven gel viscosity and causes the sharpness of peaks.

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Also, NPs displacement during the electrophoresis process enhances the uneven distribution of NPs. Immobilization of NPs by anchoring, grafting, and tethering to PAM gel could improve the separation efficiency. Thus, there is a need to investigate the NPs tethering material and

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immobilization techniques.

Detection. The excess amount of NPs interfered with detection system, increased the

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background of images, and reduced the S/N ratio of separation profiles. The excess amount of nanomaterials could deteriorate the separation process. On the other hand, excess amounts of nanomaterial loading can elevate the diffusion. Also, nanomaterial overloading can induce uncontrolled interactions of nanomaterials with analytes and disturb the separation process. Also, black-colored nanomaterials such as CNTs may interfere with electrophoresis UV or fluorescence detection and staining methods. To address this interference, Xu and Li used a non-

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optical capacitively-coupled contactless conductivity detection technique C4D for electrophoresis of DNA samples [130]. Toxicity. SQDs were applied for detection purposes but the potential toxicity of heavy metal ions

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(e.g., Cd ions) limits their application, for instance, CdSe SQDs without a ZnS shell are toxic to liver cells after exposure to UV light [131]. UV gel documentation systems are used for detection of DNA fragments and can enhance the toxicity to SQDs. Also, interaction of SQDs with highly

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toxic staining ethidium bromide must be considered and investigated in separation of DNA

samples. In contrast with SQDs, carbon-based fluorescent nanodots such as CQDs, GQDs, and

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CNDs are potential candidates for fluorescent biodetection, due to the possible excretion from body, and ultra-low cytotoxicity at concentrations suitable for fluorescent imaging [113].

5. Conclusions and Future Outlook

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This review has highlighted some of the major advances of using nanomaterials in slab gel electrophoresis. Nanomaterials are widely used in various fields of analytical chemistry. The development of new analytical techniques based on nanotechnology has been a great success.

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The use of various form of nanomaterials such as NPs, nanotubes, nanorods, nanowires, NCs,

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nanodiamonds and nanodots has led to the creation of new powerful tools applicable in different areas of the analytical chemistry. Efficient synthesis and functionalization methods elevate the application of NPs in various analytical methods including CE, MCE, CEC, LC, GC, and lab-ona-chip systems.

Nanomaterials could improve the analytical figures of merit by improving detection, selectivity, and efficiency. Nanomaterials with high thermal conductivity and low electrical conductivity are unrivaled candidates for improving the thermal conductivity of polymer

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hydrogels. Also, immobilization of nanomaterials on polymer networks could improve the separation efficiency. Uniform distribution of NPs in polymer matrix enhances the separation process by removing concentration and viscosity gradient. NPs anchoring to the polymer surface

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could stabilize the NPs in hydrogel matrix. Then, anchoring, grafting, and tethering of

nanomaterials on polymer hydrogel will be the future trend for enhancing the efficiency of slab gel electrophoresis. Also, semiconductor and carbon-based dots showed novel detection

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capabilities towards proteins. Direct in-gel detection of proteins after PAGE can be easily

obtained by SQD, CND-based fluorescence imaging method. SQD, CND-based fluorescence

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imaging methods are fast, high resolution, more sensitive, resistant to photo bleaching and photoor chemical degradation, compared with other staining methods. SQD, CND-based fluorescence imaging methods could be useful for the detection of low-abundance proteins. Novel nanomaterials such as nanocellulose can be a promising material for application in gel

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electrophoresis owing to its biocompatibility, biodegradability, and physical characteristics [132]. Nanocellulose can form highly porous films, papers, or gels with enhanced mechanical, optical, and thermal properties. For example, nanocelloulose-epoxy resins showed excellent

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thermal conductivity compared with conventional resins [133], which can be used for reduction of Joule heating effects and band broadening in gel electrophoresis. Recently, nanocellulose

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crystals used for fabrication of organic-inorganic hybrid capillary column for high efficiency enantiomer separation by open-tubular capillary electrochromatography (OT-CEC) [134]. Also, sulfonated nanocellulose used as sorbent materials in efficient, environmentally friendly dispersive micro solid-phase extraction (D-µSPE) CE method for determination of Ag NPs [135]. Boron nitride nanotubes (BNNTs) are another interesting materials to enhance the mechanical and thermal properties of polymer hydrogels. BNNTs display high thermal

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conductivity (~ 350 Wm-1K-1), owing to their unique structure and heat conducting nature of boron nitride crystals. Also, BNNTs show low electrical conductivity (~ 6 eV) which is beneficial for application in gel electrophoresis [136]. Further, hybrid organic-inorganic NCs

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(e.g. graphene oxide/nucleic-acid-stabilized silver NCs) with remarkable fluorescent properties and photostability [137] and hybrid organic-inorganic SQDs (e.g. BSA and AgInS2-ZnS SQDs) [138] can be the next generation of nanomaterials for application in slab gel electrophoresis. In

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the next decades, advances of NP modified separation media will be required in efficiency,

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reproducibility, mechanical-thermal stability, detection, sensitivity, and manipulation.

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List of Abbreviations Used BSA = bovine serum albumin CE = capillary electrophoresis

CNT = carbon nanotube CRP = chronic inflammatory CNDs = carbon nanodots CQDs = carbon quantum dots DHPC = 1,2-dihexanoyl-sn-glycero-3-phosphocholine

ED = electrochemical detection

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DMPC= dimyristoyl-sn-glycero-3-phosphocholine

EMSA = electrophoretic mobility shift assay GC = gas chromatography GCN = graphitic carbon nitride GNP = gold nanoparticle GQDs = graphene quantum dots

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CEC = capillary electrochromatography

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HEC = hydroxyethylcellulose

HPC = hydroxypropylcellulose

HPLC = high pressure liquid chromatography

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HPMC = hydroxypropylmethylcellulose LC = liquid cheomatography MC = methylcellulose

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MCE = microchip electrophoresis

MGE = microchip gel electrophoresis MMT = montmorillonite

MNP = magnetic nanoparticle MOF = metal organic framework MWNT = multi-walled carbon nanotube NC = nanoclusters NP = nanoparticle

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PAES = poly (arylene ether sulfone) PAGE = polyacrylamide gel electrophoresis PAM = polyacrylamide PDADMAC = polydiallyldimethylammonium chloride

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PDMA= polydimethylacrylamide PDMAM = poly (N,N-dimethylacrylamide) PEG-DA = poly (ethylene glycol) diacrylate PEO = polyethylene oxide

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PEO-PPO-PEO = polyethylene oxide-polypropylene oxide-polyethylene oxide PNIPAM = poly (N-isopropylacrylamide)

PVA = poly (vinyl alcohol) PVP = polyvinylpyrrolidone SWNT = single-walled carbon nanotube SQDs = semiconductor quantum dots

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TEMED = tetramethylethylenediamine

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PSP = pseudostationary phase

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Nanocomposite polymer hydrogels enhanced the fluorescence detection of proteins.

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Nanomaterials could improve the analytical figures of merit by improving detection,

•

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selectivity, and efficiency.

SQD and CND-based fluorescence imaging method could be useful for the detection of low-abundance proteins.

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The uniform distribution of NPs in gel reduced the concentration gradient and

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increased the signal intensity.

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