Temperature and Denaturing Substances Influence on Lab-on-a-Chip Electrophoresis

Temperature and Denaturing Substances Influence on Lab-on-a-Chip Electrophoresis Qualitative and quantitative determination of proteins in different biological fluids is of great significance in medicine, due to their importance in diagnosis and treatment of some diseases. Nowadays, different methods for protein analysis are available. Lab-on-a-chip electrophoresis is a relatively new technique, based on microfluidics, which allows samples of biological fluids to be analyzed within a microchip. This paper describes the optimization of performance of the chip-based protein analyses in serum samples from patients with different neurological disorders. Using microchip technology, serum proteins with the molecular mass from 4.5 to 240 Kb were separated and sized. The fluorescence detection method in the analysis was used to follow the influence of the temperature and the type and concentration of denaturing substances on the electrophoresis protein profiles. It was noted that, depending on incubation temperature and denaturing substances, different electrophoresis patterns can be obtained from the proteins of one specimen. Significant change of the fluorescence intensity was observed when different incubation temperatures were used, probably due to fluorescence quenching. In some cases, the band intensity was changed several times. Lab-on-a-chip electrophoresis is a very efficient method for the separation and determination of different serum proteins in a very short time. However, to obtain comparable results for the analysis, the denaturing agent concentration and temperature must be observed and maintained carefully. Uticaj Temperature I Sredstava ZA Denaturisanje NA Elektroforezu Vrste >Laboratorija NA Čipu< Zbog važnosti proteina u dijagnostici i lečenju nekih bolesti, njihovo kvalitativno i kvantitativno određivanje u biološkim tečnostima od velikog je značaja u medicini. Postoje različite metode za analizu proteina. Elektroforeza vrste >laboratorija na čipu< relativno je nova tehnika, zasnovana na mikrofluidici, što omogućava analiziranje uzoraka bioloških tečnosti u okviru mikročipa. U radu je opisana optimizacija postupka analize proteina na bazi čipa u uzorcima seruma pacijenata sa različitim neurološkim poremećajima. Pomoću tehnologije mikročipova, razdvojeni su i izmereni proteini iz seruma sa molekulskom masom 4,5-240 Kb. Korišćenjem pri analizi metode fluorescentne detekcije praćen je uticaj temperature i tipa i koncentracije sredstava za denaturisanje na elektroforetske profile proteina. Uočeno je da se, u zavisnosti od temperature inkubacije i sredstava za denaturisanje, od proteina istog primerka mogu dobiti različiti elektroforetski obrasci. Primećena je i značajna promena intenziteta fluorescencije pri različitim temperaturama inkubacije, verovatno usled gašenja fluorescencije. U nekim slučajevima je po nekoliko puta menjan intenzitet trake. Elektroforeza vrste >laboratorija na čipu< je vrlo efikasan metod za separaciju i određivanje različitih proteina u serumu u veoma kratkom vremenskom roku. Međutim, moraju se pažljivo posmatrati i održavati koncentracija i temperatura sredstva za denaturisanje, da bi se dobili uporedivi rezultati za analizu.

Klju~ne re~i: elektroforeza, ga{enje fluorescencije, »laboratorija na ~ipu«, neurolo{ka oboljenja, analiza proteina, uzorci seruma Summary: Qualitative and quantitative determination of proteins in different biological fluids is of great significance in medicine, due to their importance in diagnosis and treatment of some diseases. Nowadays, different methods for protein analysis are available. Lab-on-a-chip electro phoresis is a relatively new technique, based on microflui d ics, which allows samples of biological fluids to be analy zed within a microchip. This paper describes the optimization of performance of the chip-based protein analyses in serum samples from patients with different neurological disorders. Using microchip technology, serum proteins with the molecular mass from 4.5 to 240 Kb were separated and sized. The fluorescence detection method in the analy sis was used to follow the influence of the temperature and the type and concentration of denaturing substances on the electrophoresis protein profiles. It was noted that, de pending on incubation temperature and denaturing substances, different electrophoresis patterns can be obtained from the proteins of one specimen. Significant change of the fluorescence intensity was observed when different incubation temperatures were used, probably due to fluorescence quenching. In some cases, the band intensity was changed several times. Lab-on-a-chip electrophore sis is a very efficient method for the separation and determi nation of different serum proteins in a very short time. However, to obtain comparable results for the analysis, the denaturing agent concentration and temperature must be observed and maintained carefully.
Keywords: electrophoresis, fluorescence quenching, lab-ona-chip, neurological diseases, protein analysis, serum samples and disorders in the function of the blood-brain barrier. Conventional electrophoresis methods, mainly sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and disc-PAGE are widely used in performing the protein profiling (1). However, since a rapid and precise method is needed, improvements of the techniques are daily investigated and included in the analysis process.
Lab-on-a-chip electrophoresis is a relatively new improved technique that can perform protein profi ling within the confines of a microchip. This technique inte grates several procedures, such as sample handling, separation, staining, destaining, detection and ana lysis, all in a single process (2)(3)(4). Miniaturizing the laboratory onto a chip is considered to be a step for ward, due to the number of advantages, mostly in the time and labor frame of work. It requires smaller amounts of material, therefore conserving both sam ple and reagents, which is important in clinical analyses where sample quantities are often limited. The small scale of the chip increases the speed with which samples are processed and also, since the manual work is limited in comparison to conventional electrophoresis, results are more accurate and reproducible.
Optimization of the conditions for handling samples is of great importance when clinical appli cation of the technique is required. One of the basic parameters that should be optimized, especially when proteins are considered, is the incubation tem pe ra ture and dena tu ring substances. Proteins show diffe rent bonding affi ni ty to the denaturing agents when incubated at different temperatures. Despite of the most abandoned denatu ring reagent used, so dium dode cylsulphate (SDS), addi tional agents can be used, such as b-merca pto ethanol (BME) and urea. Their concentration differs in relation to the sample protein concen tration and the temperature of incubation. These denaturing agents, as well as temperature increases lead to changes in the fluorescence detection signal (5).
Due to the mentioned requirements, and in the process of achieving better and faster protein profiling, we have optimized and studied these parameters and their influence on the patients' serum protein profiles using lab-on-a-chip electrophoresis.

Materials
Serum samples were obtained from the bio chemical laboratory in the Clinic of Neurology (Faculty of Medicine), and were labeled according to the Declaration of Helsinki ethical committee. High molecular mass (HMW) and low molecular mass (LMW) protein standards were obtained from BioRad Laboratories. Denaturing agents, SDS, b-mercaptoethanol (BME) and urea were obtained from Sigma-Aldrich. Lab ona-chip electrophoresis was carried out on the Agilent 2100 Bioanalyzer from Agilent Techno logies, Santa Clara, USA. Protein 200 Plus LabChip kit was used in the analysis.

Methods
Serum samples were collected from whole blood by vein punction, and stored at -80 °C until analyzed. Before handling the serum samples, total protein, albumin and immunoglobulin G (IgG) concentrations need to be measured. The total protein concentration in the serum was determined by a spe ctrophotometric method, using the Alcyon 2100 Bio ana lyzer. Turbidimetry was used to analyze albumin and immunoglobulin according to the prescript procedure. All chips were prepared accor ding to the Agilent protocol provided. The samples were set using the proposed procedure, as well as the opti mized procedure. The gel and the gel dye mix were prepared as proscribed in the Agilent manual (5,6).
When optimization of the denaturing agent was performed, samples were prepared using 79 μL deionized water, 4 μL sample and 7 μL of BME. Protein standard was included in every run, therefore providing comparison between different runs. The influ ence of the additional denaturing agents (SDS and urea) was determined using the same procedure of sample preparation, and the signals of the proteins were observed. Afterwards, different concentration of the denaturing agent was used in order to obtain the optimal sample: denaturing agent ratio. When the tem pe rature influence was studied, sample incubation was done at five different temperatures: 5 °C, 22°C (room temperature), 37°C, 60°C and 90°C. After sample preparation, samples were loaded onto a chip. The chip was placed in the bioanalyzer and run. In the process of electrophoretic separation in the bio analyzer, the electric current provides ideal conditions for staining and destaining within 45 s per sample. The analysis chamber itself is heated to 30 °C to ensure the constant conditions. After the separation, the signals are detected by laser-induced fluoresce nce detection (670-700 nm) (6-8). The Protein 200 Plus software assay was used to obtain the complete numerical analysis. The reproducibility and statistical analysis were performed using Statsoft Statistica v7.0.61.0.

Results and Discussion
Lab-on-a-chip analyses have proven to be more precise and less troubleshooting when analyzing complicated protein mixtures, in comparison to con ventional electrophoresis. The analyses of serum samples from the patients were performed in order to determine their protein profile. Both control group samples and patient samples were analyzed and compared to the protein standard (Figure 1).
Determination of the protein molecular mass was done, as well as band-shape and peak analysis. Protein sizing with the chip-based analysis was performed by running a protein standard consisting of 10 proteins (HMW plus LMW standard in optimal ratio) and was analyzed in the terms of examining reproducibility (data not presented). Results have shown that the protein profile differs in samples with neurological diseases in the range of appearance of different additional protein fractions. Concentration changes have also been noted for the immuno globulin fractions in the patients suffering from system disorders, and for the alpha-globulin fraction in patients with inflammation diseases.
We used this method to analyze the influence of several denaturing agents and their concentration on the sample preparation. The effects on the electrophoretic protein profile were observed and explained using the method of laser-fluorescence quenching. The samples were prepared under reducing con ditions, using SDS, urea and BME as reducing agents. It was noted that incubation with BME gives best resolution ( Figure 2).
Further, BME concentration in the sample (total protein: denaturing agent) was optimized. We have used different concentrations varying in ratios TP: BME (1:1; 1:1.5; 1:2; 1:2.5: 1:3; 1:3.5 and 1:5). The results have shown that the optimal ratio for serum sample analysis is 1:3.5 ( Figure 3). Additional increase in the BME concentration does not influence the resolution and signal of protein peaks.  It is noted that the fluorescence signal increases when the denaturing agent concentration increases to a certain level. This effect can be explained when the fluorescence mechanism is considered. In the native state, the fluorescence from the dye molecules is severely quenched. When proteins are denatured they unfold, and the fluorescence increases. Such a dra matic change is measurable even at the single molecule level, permitting the study of folding dyna mics of protein molecules in real time (8,9).
When analyzing protein mixtures, temperature is a very important parameter due to its effects onto the protein conformation and affinity to bind with the dye, and therefore to increase the signal of detection. For providing denaturing conditions, sample preparation should be done by boiling with the denaturing agent. In Disc-PAGE, however, it is noted that lower tempera tures provide better results. We have analyzed this effect at 5 temperatures of incubation using the opti mized denaturing agent, BME, in the optimal concen tration within sample preparation. It was noted that the optimal temperature for generating denaturing conditions is 90 °C (Figure 4).
Significant changes in the peak intensity were observed. The effect is similar to when denaturing agents are considered. Because of the samples' complicated structure and the three-dimensional structure of the protein constituents, the dye molecules are in close proximity with each other and their fluorescence is severely quenched. When heated and denatured, proteins unfold, swell and reduce the proximity among dye molecules, thus leading to an increase of the fluorescence.
Lab-on-a-chip is more efficient technique when analyzing complicated protein mixtures in comparison to conventional electrophoresis. However, its usage in routine analyses is still limited, due to the need of an optimized method which is easy to handle, cheap and follows certain procedure. The serum proteins are a complicated mixture and are very sensitive to changes in sample temperature and denaturing agents. Significant change of the fluorescence intensity is observed probably due to fluorescence quenching. In some cases, the band intensity was changed several times. Therefore, further analyses are necessary in order to ensure implementation of the technique in routine laboratory practice. Relative absorbance / (FU)