As we all know, science has a great impact on our daily life. Through science we can learn and understand our surrounding. We can use the knowledge gained through time to improve our lifestyle by making advances on health and technology.  In other words, science gives us the proper answers that make easier the understanding of the world’s mysteries.

To study these mysterious areas we require more scientists and science employees that are willing to dedicate their lifes trying to discover significant findings to improve our lifestyle. To achieve this purpose it is necessary to have knowledge, dedication and most importantly commitment; but it’s crucial to have successful research experiences.

BioMinds Program brings me an extraordinary opportunity, as researcher, to improve my academic potential and demonstrate all the knowledge gained through my undergraduate education.  This Program helps students to decide if they want to achieve a PHD and expend their life in laboratories with the purpose of find innovative information and/or discover amazing things; or try to go straight into the world of work after completing their Bachelor Degree.

In addition, this extraordinary program improve my perseverance and patience helping me to keep trying to achieve our research objectivities even though the experimental data collected was not expected or the procedure was not complete as we desired. Also help me to improve my abilities relating to the work in group knowing that is an expected skill in every employee.  To conclude, I thank Biominds Program to gave me the chance to grow as a student but specially for the opportunity to grow as a professional.

summarized in two words: keep trying!

As I mentioned in my previous posts, my lab partners, mentors and I are working with the development of a biosensor capable to detect hydrogen sulfide (H2S) efficiently.  The interest in the detection of H2S arises from the detrimental effects that exposure to this gas: health, environment, and industrial facilities care. To accomplish it we use different electro analytics techniques. The two techniques more important are cyclic voltammetry and ampereometry studies. Previously posts present some of the results that we obtained.  These results demonstrate that several of our goals were accomplished, but despite this, we were trying to achieve our more crucial goal. We want to compare our results with the results obtained (under the same conditions) with the more efficient instrument existing, Apollo 4000 Free Radical Analyzer Electrode.  Apollo is a instrument  that detect our interested compound but it leads a tedious procedure that involve the difficulties when the membranes will replace, the repeated breaking of membranes and the complications presented at the moment of fill correctly the sleeve with a cyanide solution such that the solution makes contact with the membrane. Other disadvantage is that Apollo can’t detect low concentrations of hydrogen sulfide.

In  the post “science without discoveries is like a book without words…” published on November 22, 2010, I presented the results of the same studies that we are supposed to do with Apollo, but in this case using our modified electrodes. The ampereometry results demonstrate that the linkers made possible the detection of low concentrations of H2S. The calibration curve evidences that using our electrode, a concentration of H2S can be estimate according of the current value obtained by the sensor.

These results are very significant in our research but they do not say much without a rigorous comparison with Apollo’s results. This is why we spent various months trying to replace the membrane correctly and avoiding its rupture. During a long time the efficient replace was impossible. We read again and again the manual of the equipment, asked for help to people who became familiar with it, trying to figure out new ways to carry out the filling, but nothing worked. Finally this week we could did it, finding that was affecting the procedure. Our mentor turned the electrode by mistake causing, as consequence of its pH, that the solution adhere to the membrane. Then we leave the electrode over 12 hours polarizing and after it the electrode was ready to use. Our final results present that is true that Apollo detect H2S concentration but, at difference of our electrode, it can’t read relatively low concentrations and its use is rather tedious.

As Biominds program requirement, all participants must present their project on 31^st Puerto Rico Interdisciplinary Scientific Meeting & 46^th Junior Technical Meeting” (PRISM/JTM 2011) in the Inter American University at Bayamon, Puerto Rico on Saturday March 12, 2011. Below I present the abstract that summarizes my Research (Development of a Novel Biosensor Using of Lucina pectinata Hb I and Its Characterization by Electroanalytical Technique) and I will be presenting it with my partner Natalia Vazquez.

ABSTRACT:

Lucina pectinata is a clam present in the southwest coast of Puerto Rico and has a hemoglobin (HbI) that is involved in the transportation of H2S to symbiotic bacteria. The interaction and extraordinary affinity of HbI to H2S allows us to propose the preparation of a hydrogen sulfide sensor for the characterization of this gas in aqueous systems. Electrochemical analysis of H2S was done on a modified gold electrode with cysteine and Ni2+ ion attached to recombined Hemoglobin I from Lucina pectinata (rHb I). Cyclic Voltammetry (CV) was used to characterize the entire modification process as well as the electrochemical response of H2S amounts added to the system. In addition, DC Potential Amperometry technique was used to quantify H2S through the construction of calibration curves based on the current change when increases the concentration of H2S. Results support the possibility of constructing a sensor for the characterization of H2S by this assembly.

~by Keren Valentin

Like I had explained previously, our research focuses in the development of a novel biosensor capable to detect efficiently the H₂S concentration.  The reason that motivated to us to develop the mentioned biosensor is that at present, an instrument capable to detect quantitatively the concentration of H₂S with exactitude and precision (much less in a short time interval) doesn’t exist. To achieve it, we modified gold electrodes with recombine hemoglobin of Lucina pectinata (rHBI) and cystein. Electrodes were study by different electroanalytical procedures like cyclic voltammetry (to modified electrodes) and ampereommetry (to determine the way in which the concentration of Na2S affects the current produce by the redox reaction). Figure 1 present the electrode modification.

I know that you as a reader asked yourself: What kind of benefits we can obtain detecting H₂S concentrations? Studies demonstrate that some illness and others health disorders, volcanic eruptions and environmental problems (contamination) occur as consequences of higher or low concentration of H₂S but wasn’t possible to measure or detect this concentration. Also is very useful the development of this kind of biosensor because in recent years, interest has been directed towards H₂S because has been  shown to exhibit potent vasodilator activity both in vitro and in vivo.

Figure 2 presents the results obtained by the amperometric titration of the modified electrode (Au/Cys/Ni+2/rHBI). This technique is useful because offers quantifiable information about how the current change as consequence of the concentration of Na2S. The equipment used was: Epsilon electrochemical analyzer, Working Electrode (Au/Cys/Ni²⁺/rHbI), Reference Electrode (Ag/AgCl sat. NaCl 3M) and Counter Electrode (Pt wire).  The experimental process takes place in a medium with a pH of approximately 6.7. The buffer used was Phosphate.  The use of a correct buffer with a specific pH is very important. One of the reasons (and the one easier to understand) is that H2S is very toxic in pH smaller than 6.5. Obviously work with a gas sample avoiding losing it is very complicated and make quantitative additions is some difficult. For this reason we used Na2S to make the ampereometric analyze knowing the 1:1 relation between Na2S and H2S. Specific micro-concentrations of Na2S are added to the buffer using injections or micropipettes. It reacts with water producing H2S (Figura 3).

Figure 3

Is very important to note (in the Figure 2) the change s in current by the addition of Na₂S demonstrating at the same time that the modified electrode response to micromolar concentrations of Na₂S. Finally this technique permit to us identify at what concentration of H2S the modified electrode become saturated with the gas. Using the Faraday’s law we related the data collected in the amperometric analysis with the concentration of Na2S (Figura 4). This calibration curve shows a linear behavior between changes in currents with concentrations of Na₂S (and using the 1:1 relation between Na2S with H2S we can know the relation of current with H2S concentration). The graph presents that concentration is inversely proportional to the current. This behavior tells us that H2S is saturating the modified electrode and forming a complex with rHbI. The complex does not contribute to the current.

To conclude, we have sufficient useful information that help us to construct the desired biosensor. The next step is to compare our results with the results generated by other commercial sensor called Apollo. Apollo is one of the most used biosensor capable of detected H2S. Despite this, Apollo is not very efficient apparatus having some disadvantages. Some of the disadvantages are that only can detect low concentrations of    H2S and the solution cannot be contaminated with other substances or ions. These two disadvantages will be study using our biosensor and we have high probabilities that our biosensor work efficiently under both of them.  We also want to test it with environmental and physiological samples.  Without doubts, we know that we will get better results than expected and after a hard work will achieve our objectives impacting the world of science.

Post # 2   Aug-Dec 2010 ___________________________________________________________________________________________________________

Nashicel J. Rodríguez, student of University of Puerto Rico (Cayey Campus), is working ina bioorganic research that involves the study of a plant used in the treatment of Diabetes mellitus. As all we know, the treatments to control diabetes produce several harmful effects.  The uses of natural methods, for example different plants, are one manner to reduce these negative effects. For this reason, Nashicel is working in a interesting research were the general focus is the study  of the bioactive component present in the Agave Americana’s (maguey) infusions and crude extracts. In this case, she is analyzing and determining the bioactive structure present in the roots of Maguey. At the beginning she used the column chromatography technique, but then she used the derivatization technique and extraction process to determine if similar compounds can be extracted in a simple way. The mentioned technique consists in the breaking of a bond that connects the sugar part of the saponins with its aglycone structure. Finally, this semester she is going to continue the study of the Maguey roots’ infusion and crude extract but now she will try to identify and characterizes the potencial bioactive compounds in the mentioned Maguey roots.

Melanie Torres study Cellular Molecular Biology in the University of Puerto Rico, Río Piedras Campus. She began participating in Biominds Program (August 2009)  working in a research focuses on the molecular aspects of regeneration, specifically on the expression of genes involved in the “Ubiquitin Proteasome System” during the process of intestine regeneration of the Holothuria glaberrima. Then, after the apparition of diverse problems, she decided to change her research and began a new project involving the sequencing of the cadherin gene expressed in a normal intestine of the Holothuria glaberrima.  To achieve it she used the RT-PCR technique that involves electrophoresis, cultivation of bacteria, isolation of small plasmid DNA from bacteria (mini-prep), purification of DNA fragments from PCR reaction, digitalizing electrophoresis results and measuring DNA concentration with the “Nano Drop”.

This semester, she returns to work with her first research related with the used of sea cucumber Holothuria glaberrima as a model system to study different aspects of organogenesis and regeneration; but in this moment she is working with the characterization and the study of the gene expression of retinol dehydrogenase during different periods of intestinal regeneration of the sea cucumber. Retinol dehydrogenase is an enzyme that catalyzes the first step of the synthesis of retinoic acid from retinol.

To conclude, both researches are very amazing and I consider both of them very important in the medicine world. Finally I hope that both students can obtain excellent results at the end of this semester because I know that with this information they will be able to improve the lives of many people and in some cases until saving lives.

(Post # 1 Sept-Dec 2010)

(3^rd semester participating in Biominds Program)

This semester I have another opportunity to work in a Chemical Research under the supervision of Dr. Carmen Amarilis Vega (mentor) and the Instructor Mario Ortega. The crucial objective is to study the electroactivity of HBI for the development of a biosensor capable to identify and quantify directly H₂S.  In the past academic year significant results were achieved. We could obtained information about the stability of the equilibrium of the protein; determined the amount of electrons that are transferred during the redox reaction and modified gold electrodes using cysteine and 3-mercaptopropionic acid along with nickel ion and the recombine protein HbI (rHbI). Actually we are going to continue studying the modified electrode using cysteine.

Some of our goals for this semester are:

1. Determine a constant potential using cyclic voltammetry and then, using a calibration curve (ΔI vs. Δ[H₂S]), determine the lower detection limit in a buffer with pH = 6.7
2. Determine the interferences produced by ions in a solution containing H₂S.
3. Measure the concentration of H₂S in physiological and environmental systems.
4. Finally, we will compare our modified gold electrode with a commercial equipment called Apollo.

In order to accomplish this we will use a new technique  called “amperometric method” and also we are continuing using the cyclic voltammetry technique as did last semesters. The amperometric method involves the change of the current according to the change of concentration, under a constant potential.

Read different posts in other students’ bio-blogs is a good way to expand our knowledge in diverse areas and it teaches us definitively that the field of science is very immense and diverse. Below, I presented some important details about other students’ bio-blogs.

Paola Berrios Hildamarie Caceres is a student of University of Puerto Rico at Mayaguez. She is working with the research project: The expression and identification of FAP1 in yeast Saccharomyces cerevicae. The technique used to achieve her project objectives is SDS-PAGE. This technique consists of a vertical electrophoresis of poliacrilamida in which the proteins migrate through the gel due their molecular weight. This technique makes possible the characterization of FAP1 and TOR1. Finally, the mentioned technique validates the efficiency of the protocol and the expression of the protein under study.

Jean Padilla is a student of UPR (Rio Piedras Campus), who works in a Tryptophan Scanning Mutagenesis project. His main goal to this semester was complete their finals goals to finish the project. To achieve it, he generated the mRNA needed to construct a functional receptor in a oocyte using the mRNA microinjection technique.  Unfortunately the process of his research was 1 in a of 1-5 as a consequence of some problems with the bacteria.

Mayrim Bernand is working with the studies of the structure and function of CC-chemokines. This research involves the identification of possible motifs in the structure of chemokines that can be similar to HIV motifs that can be useful in the pharmacological targets to treat HIV. Actually she is using bioinformatics techniques. The most equivalent is Blast technique that compares biological sequence information, such as the amino-acid sequences of different proteins. In her case compare the sequences of Rantes, MIP-1α, and MIP-1β.

Talking about my research I can mentioned that we found some difficulties in order to achieve our goals. Despite this, I can expand my knowledge and have an excellent experience.  The technique that I learned this semester is the potentiometric titration. The mentioned technique permits us to obtain information about the stability of the equilibrium of the protein. Also made possible determine the amount of electrons that are transferred during the redox reaction.

Working in group always encloses various barriers and challenges. Talking about some disadvantages that all team worker have to surpass I can mention that each person of the team need to be flexible to accommodate the working hours in a way that all the researchers can work in an effective form knowing that each student has different class schedule and diverse hours available to work in the laboratory (I also presented this statement in the post # 2). The real problem is when we work directly with other persons because when one of them is not on time or damage something during the process all the team loses time retarding all the members of the research. We can only surpass this with a lot of responsibility, commitment and patient.

My Research Technique

This semester we are focuses to work with Oxidation-Reduction Potential Titration. This is an easy method that permits us to obtain a relation between the concentrations and the strength of a species to gain or lose an electron. But in our case it result difficult because we need to find the adequate concentration of the titrant in order to study the transfer of the electron referent to the iron present in each proteins. This is very important because we need to determine the perfect concentration of the tritant in order to use the less possible quantity of the HB-1 because is an expensive protein and the effectiveness of the biosensor that will be created needs to use the minimum necessary quantity of the protein.  Using a scale from 1-5 to indicate the progress that we have reached in relation to the goals that I stated in my previous bio-blog a can use a value of 2 (some progress, I have achieved at least 10% of the proposed objectives).  It is a fact that the process that we are using is not very difficult in comparison with the previously used but this method takes up a lot time because the occur very slowly. This provoke that each titrations requires a lot time to accomplish it.  In addition we are waiting to receive important necessary materials and at the moment when we received it we know that we can achieve our mentioned objectives or goals.  To overcome our goals, our instructor, divides our research group in small groups to have more period with student working in the laboratory. This permits us to increase the number of titrations done during a week.

This semester I have another opportunity to work in a Chemical Research under the supervision of Dr. Carmen Amarilis Vega (mentor) and the Instructor Mario Ortega. The crucial objective is to study the electroactivity of HBI for the development of a biosensor capable to identify and quantify directly H₂S.  Significant results were obtained, the past semester, after study the behavior of the proteins in Au electrodes modify with nafion and 3-mercatopropionic acid demonstrating that the modification improve the resultant signal.

Actually, we are going to work with the same proteins but now we will analyzed them making use of potentiometric studies. Also, we are going to prepare an efficient procedure to convert the iron present naturally in the proteins (Fe ³+) to Fe ²+ avoiding significant variation in the composition of each protein. Obviously the equipment correct managing is very important; for this reason we are going to spend several hours of job studying and practicing with the equipment.

To conclude, this semester offers me, once again, the opportunity to improve my knowledge and reinforce interpersonal relationships and the capacity to manage conflicts. Also, I am going to reinforce my abilities using diferrent equipment and experimental process like the potentiometric studies.

Visiting different bio-blogs is an excellent way to expand our knowledge in diverse areas and it teaches us definitively that the field of science is very immense and diverse. Below, I presented some important details about other students’ bio-blogs.

Hildamarie Caceres is a student that works with microorganisms founded in the salines of Cabo Rojo with the purpose to find specific enzyme activity to identify the microorganisms that present high wenzymatic activity for lipases, esterase sans proteases to finally determine their stability in organic solvents to find the right medium to grow them. Hildamarie’s work experience was very amazing because in approximately eight months she learned a lot of important and interesting things. Some of them are: cultivate microorganisms, aseptic techniques, how to use UV and Visible Light spectrophotometers, how to use a Gas Chromatographer and other important things that in my personal case I had to take various university courses (Chemical General 1 and 2, Organic and Analytical Chemistry, Physical Chemestry, etc.) to learn how to use some of this equipment. Definitively she managed her working hours very useful.

Ulises Marrero is a student, who works with proteins (HB-II), the same as me. Was very surprising that he works with Lopez Garriga (his mentor) because indirectly is my partner, the instructor and me had the opportunity to work with him too. I liked to know the fact that a lot of researchers can work and study, at the same time, with similar things or with the same variables of investigation (in this case with hemoglobin of Lucina Pectinata) but when we read the objectives and/or purposes of the research we note that they are totally different. The goal of Ulises’ research is to find optimum crystallization parameters for Lucina Pectinata (HB-II) and the formations of a Big HB crystals.

Yiaslin Ruiz works in an interesting research. She was studying the PbSe Quantum with the goal of synthesize them in different sizes and shapes to apply them in applications as solar cells and biological labeling. Like Chemical Engineering student, I consider very interesting the study of nanoparticles or nanostructures a recognized that she is participating in an excellent research.

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