Information

1.9: Biomolecule Detection - Biology

1.9: Biomolecule Detection - Biology


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Learning Objectives

Goals:

  • Employ indicators to discover characteristics of a solution.
  • Use indicators to determine contents of an unknown solution.
  • Employing positive and negative controls to validate a test.

Student Learning Outcomes:

Upon completion of this lab, students will be able to:

  • Describe the properties of some important biomolecules.
  • Explain important characteristics of proteins and carbohydrates.
  • Perform tests to detect the presence of carbohydrates and proteins.
  • Explain the importance of a control in biochemical tests.
  • Use a biochemical test to identify the presence of a molecule in an unknown solution.

INTRODUCTION

The Macromolecules of Life: Proteins, Carbohydrates, and Lipids

The cells of living organisms are composed of large molecules (macromolecules) sometimes also referred to as organic molecules because of the presence of the element carbon. Very many of the organic molecules found in living organisms are carbohydrates, proteins, lipids, and nucleic acids. Each of these macromolecules is made of smaller subunits. The different molecules have different chemical properties. For example, monosaccharides such as glucose will react with a chemical agent called Benedict’s solution but disaccharides, like sucrose, and polysaccharides, like starch will not. Similarly, proteins will react with a mix of potassium hydroxide and copper sulfate but free amino acids, carbohydrates, and lipids will not.

Today, we will focus on three of these molecular types: lipids, proteins and carbohydrates. You will work with nucleic acids in another lab. You may want to review the properties of the biomolecules of life.

Figure 1: The molecular and macro structures of sucrose, starch, lipids, and proteins.
Molecular Type

Molecular Structure

Macro Structure

Sucrose

Common source: Table sugar

Starch

Common source: Rice

Lipids

Common source: Cooking oils

Proteins

Common sources: cell receptors, egg, hair, feathers

Part I: Controlled Experiments to Identify Organic Compounds

Indicators are chemicals that change color when chemical conditions change, such as pH, or when a chemical reaction takes place producing a colored molecule. There are many biochemical procedures that can be used to detect the presence of important molecules. In this exercise, you will test various solutions in order to detect the presence of these molecules. We will employ controls as we test the solutions. Controls provide results to compare to the solution being tested. Controls should give predictable results. By comparing the test solution result with the controls, you can determine the result of the test solution.

A positive control contains the variable for which you are testing. When the positive control is tested, it reacts in an expected manner. If, for example, you are testing for a type of carbohydrate in unknown solutions, then an appropriate positive control is a solution known to contain that type of carbohydrate. The resulting reaction, when properly performed, will demonstrate that the reagents work as expected and shows what the result should look like if the test solution is positive. If the positive control does not react as expected, your test is not valid. Perhaps your test reagents are not working properly.

A negative control does not contain the variable for which you are testing. Often a negative control contains only water. It will not react with the indicator reagents. Like the positive control, the negative control solution shows you what a negative result looks like and verifies that the detecting reagent is working properly. If the negative control does react, your test result is not valid. Perhaps the control solution or reaction tube was contaminated with the test variable.

I. Carbohydrates

Benedict’s Test for Monosaccharides

Molecules made of the atoms carbon (C), hydrogen (H), and oxygen (O), in a ratio of 1:2:1 are carbohydrates. For example, glucose, one of the most important carbohydrates for living cells, has the chemical formula C6H12O6. Simple sugars also known as monosaccharides are carbohydrates. Paired monosaccharides form disaccharides. A common example of a disaccharide is the table sugar, sucrose. It is composed of the monosaccharides glucose and fructose linked to fructose. Similarly, linking three or more monosaccharides forms a polysaccharide. Starch, glycogen, or cellulose are polysaccharides important to cells and have many monomers of glucose linked together in different ways.

Starch

Benedict’s reagent is the indicator we use to detect monosaccharides. When monosaccharides are mixed with Benedict’s and heated, a color change occurs. If there is a small amount of monosaccharide in the solutions, a greenish solution is produced. If the solution contains a large amount of monosaccharide, an orangish precipitate results. A precipitating solution means small particles settle out of the solution.

Reaction 1.

Monosaccharides + Benedict’s reagent + Heat ⇒ Green to Orange

II. Proteins

The cell relies on proteins for very many functional reasons. Proteins may be enzyme catalysts, form channels for molecules to pass across membranes, form structures and more. The subunit of protein molecules are monomers of amino acids. The bond that forms between amino acids to form protein is called a peptide bond.

Peptide bonds can be detected by using two chemical reagents, potassium hydroxide (KOH) and copper sulfate (CuSO4). Potassium hydroxide causes a protein to break apart so that copper sulfate can react with the peptide bonds. The resulting color is purple. The more protein, and hence more peptide bonds, in the solution, the darker the resulting purple will become.

Testing for Monosaccharides with Benedict’s Reagent

Reaction 2.

Proteins + KOH + CuSO4 ⇒ Purple

Materials
  1. Test tubes labeled with the contents you will add to each tube
  2. Beaker with water and hot plate (water heated to near boiling)
  3. Metric ruler
  4. Marker
  5. Deionized water and carbohydrate solutions
  6. Appropriate tool to remove hot tubes from water
Procedure
  1. Obtain 5 test tubes and number them 1 – 5.
  2. Use a marker to indicate 2.5 cm from the bottom and another mark at 5cm from the bottom.
  3. Fill each test tube to your 2.5 cm mark with the appropriate solution:
    1. Distilled water 2. Concentrated glucose solution 3. Diluted glucose solution 4. Sucrose solution 5. Starch solution
  4. Add Benedict’s solution to each tube to the 5 cm mark.
  5. Place all of the tubes in a hot (90°C) water-bath for 2 min, and observe color-changes during this time.
  6. After 2 min, remove the tubes from the water-bath and record the color of their contents in the table below. Also observe your classmate’s reactions.
Observations

Perform the Benedict’s test for monosaccharides. Reproduce this table in your lab book and complete it with your observations.

Data Table 2.

Tube Contents

Color after reaction

Presence of monosaccharide?

1. Water

2. Concentrated glucose

3. Diluted glucose

4. Sucrose solution

5. Starch solution

Instructions to clean up

* Clean tubes are very important. Contaminated tubes may influence results of future tests.

1. When your observations are complete, carefully wash and rinse the tubes following the instructions in part 2. You may leave the markings on them until the final clean up procedure of the day.

Data Analysis
  1. Which of the above solutions serve as your positive control? Negative control?
  2. Examine your test and your classmates test solutions. Which solutions were positive for monosaccharides?
  3. Which contains a higher concentration of monosaccharides, potato juice or onion juice? How do you know?
  1. Which solutions did not react with the Benedict’s solution?

Testing for Peptide Bonds (Protein)

Materials
  • Four clean test tubes labeled with the contents you will add to each tube
  • deionized water, and test solutions
  • Indicator reagents potassium hydroxide (KOH) and copper sulfate (CuSO4)
Procedure

Perform the Peptide Bond test for Protein

Caution!

Do not spill the KOH – it is extremely caustic. Rinse your skin if it comes in contact with KOH.

  1. Use your four clean test tubes from the previous procedure. They still need to be numbered and marked at 2.5 and 5 cm from the bottom.
  2. Fill each test tube to the 2.5 cm mark with the appropriate solutions indicated below
    1. Water
    2. Protein Solution
    3. Amino Acid Solution
    4. Test Solution
  3. Add potassium hydroxide (KOH) to the 5cm mark on each test tube.
  4. Add five drops of copper sulfate (CuSO4) to tube and mix well.
  5. Record the color of the tubes’ contents in the table below. Also observe your classmate’s reactions.
  6. When finished dump the contents of the tubes and wash them. Rinse with distilled water.
Observations

Perform the Protein Test: Reproduce this table in your lab book and complete it with your observations.

Data table 3.

Tube Contents

Color after reaction

Presence of protein?

Water

Protein solution

Amino acid solution

Unknown solution

Instructions to clean up

*Clean tubes are very important. Contaminated tubes may influence results of future tests.

When your observations are complete, carefully wash and rinse the tubes following the instructions in Part I.

Data Analysis
  1. Which of the solutions is a positive control? Which is a negative control?
  1. Do individual amino acids have peptide bonds? How do you know this to be true?
  1. What type of solution did you test as your unknown? Did it contain protein?
  1. Observe your classmates reactions and describe which unknown solutions contain the most and the least protein. How can you tell?

III. Lipids

Lipids are a class of molecules that are not soluble (do not dissolve) in water. They are composed of the molecular building blocks of glycerol and three fatty acids. Fatty acids come in two major types, saturated and unsaturated. This difference is due to the presence of particular types of bonds within the fatty acid molecule (see figure) and affect the shape and characteristics of the overall lipid containing these fatty acids. You may want a review of lipids.

Testing for Lipid with Sudan IV

Caution!

Use gloves and avoid contact with Sudan IV as it is considered a possible carcinogen. Immediately wash your skin with soap and plenty of water if you come in contact with the solution.

Materials
  • Filter paper (small enough to fit in the petri dish) and pencil with areas labeled for test substances
  • clean empty petri dish
  • solution of 0.2% Sudan IV
  • Gloves (see safety warning)
  • Dedicated transfer pipettes or micropipettes with tips.
  • Solutions of deionized water, vegetable oil, and test solutions (cream, dairy milks, coconut milk, soy milk etc.)
  • optional- hairdryer
Procedure
  1. Obtain filter paper and on the far edge mark with pencil which solutions will be placed toward the interior of the mark.
  2. Drop a small amount of solution near the appropriate mark. 1. Vegetable oil 3-6. Test solutions
  3. Allow to dry. Use a hairdryer to speed up this process.
  4. While the paper is drying, answer the Data Analysis questions below.
  5. Soak the paper in the petri dish containing 0.2% Sudan IV. (handle with gloved hands)
  6. Rinse the paper in distilled water and allow to dry.
  7. Record the color of the spots in the table below. Also observe your classmate’s reactions.
Observations

Sudan IV test for lipid: Reproduce this table in your lab book and complete it with your observations. The darker the stain, the more lipid is present.

Data table 4.

Spot Contents

Color after reaction

Relative amount of lipid?

1. Water

2. Vegetable oil

3.

4.

5.

6.

Instructions to clean up:

When your observations are complete, carefully dispose of any remaining Sudan IV solution in the container provided by your instructor. Always use gloves and do not move the container if there is a danger of spilling.

Data Analysis
  1. Which of the above solutions serve as your positive control? Your negative control?
  2. Hypothesize which solutions will contain the greatest amount of lipid. Why do you believe this to be true?
  3. Which solutions contained the greatest amount of lipid?
  4. Did your observations support your hypothesis? Were you surprised by some of the results? Explain.

Part II. The Saga of the Soda Dispenser

Enrique was a new employee. This was his first job and he had only been on the job for a couple of weeks and was still on “hiring probation.” He liked the crew he worked with and the paycheck that would come every few weeks. He wanted to stay. Today, there was a problem and he had to figure out something fast to solve it. He knew that if he did, the manager would be really pleased and his job was guaranteed.

Someone was complaining that the soda dispenser was dispensing “regular” cola from the “diet cola” dispenser. The customer claimed to be on a reduced-calorie diet and was not happy about the extra calories consumed. There was more at stake than one unhappy customer, though. The manager told Enrique that many of their customers were diabetic and consuming sugar-laden soda could alter their blood-sugar chemistry in a dangerous way. They could not allow those customers to be harmed.

Scope of the Problem

If the diet soda dispenser did have regular soda, then did the regular soda dispenser have diet? What about the Dr. Pepper dispenser? That, at least, tasted like Dr. Pepper, so it was OK- or was it? What a mess! Should they throw all the soda in the dispenser out and start again? Or was there some way of determining if the soda was being dispensed correctly? If they could determine what the problem was, they could save the business money and not waste the soda products.

Enrique’s Attempt to Solve the Mystery

Enrique knew that most soda had high fructose corn syrup in it but diet soda had sugar substitutes in it: Substitutes that were not sugar but fooled your taste buds into believing it was.

Questions for your lab book:

  1. Does the regular soda have high fructose corn syrup in it? Look at the label determine if it does or doesn’t. Write your observation in your lab book.
  2. Does the diet soda have high fructose corn syrup in it? Look at the label determine if it does or doesn’t. Write your observation in your lab book.
  3. Determine whether fructose is a monosaccharide, disaccharide or polysaccharide.
  4. Can we do a test?

Just the other day, in science lab, Enrique had run some tests on solutions in order to determine their compositions. One of the tests was for detecting monosaccharides in solution! He knew his science teacher would still be in the classroom at this time and the school was barely a 5 minute walk from the restaurant. He could solve the mystery in under 30 minutes! Enrique quickly told his manager his plan and grabbed some cups of soda, which he labeled, so he could tell which dispenser they came from, then headed out. Enrique quickly ran to the school lab and got permission to run his experiment. Help Enrique set up an experiment to test the soda.

More questions for your lab book:

  1. Would it be a good idea to include controls? If so, which solutions?
  2. Which detector reagent(s) will you use?
  3. What colors will you look for to indicate the presence of the “regular” soda?
  4. How many test tubes do you need? How will you label them?

Testing Unknown Soda Solutions

Materials
  1. Clean test tubes labeled with the contents you will add to each tube
  2. deionized water, and solutions to test
  3. Indicator
Procedure

Perform the test for monosaccharides:

  1. Obtain the needed number of clean test tubes and mark them at 2.5 and 5 cm as before. Code them as to the contents (numbers corresponding to your solutions- which you record below)
  2. Obtain the unknown solutions from your instructor.
  3. Fill the tubes to the 2.5 cm mark with the control and test substances.
  4. Fill the tubes to the 5 cm mark with indicator and treat was needed.
  5. Reproduce this table in your lab book and complete it with your observations, then answer the questions regarding the soda saga.
Observations

Perform the Appropriate Test: Reproduce this table in your lab book and complete it with your observations.

Data table 5.

Tube Contents

Color after reaction

Presence of fructose?

Diet or regular?

1.

2.

3.

4.

5.

6.

Instructions to clean up:

Caution!

DO NOT allow ethanol to come in contact with the hotplate. Ethanol is very flammable.

*Clean tubes are very important. Contaminated tubes may influence results of future tests.

  1. When your observations are complete, carefully wash and rinse the tubes following the instructions in part 1.
  2. At the end of the lab period be sure all labels are removed from the tubes using a small piece of paper towel and ethanol.

Final Conclusion

  1. What does Enrique tell his manager? Is the soda dispenser messed up or not?
  2. What, if any, soda needs to be changed?

Study Questions

  1. Why should you always include controls in each procedure?
  1. What serves as a good negative control and why?
  1. Describe a positive control.
  1. If you run a test for monosaccharide on what you believe is “regular” lemon lime-flavored soda, but the solution is sky-blue after heating with Benedict’s what does this tell you?
  1. What if only AFTER running your test, you read the label of the lemon-lime soda and notice that the ingredients do not contain fructose but does contain sucrose. Is your test procedure faulty or is there another explanation for your result?

Attributions

  1. Sucrose Molecular Structure from 5.2 Carbohydrates.
  2. Protein Structure diagram by Lady of Hats, Public Domain, via Wikimedia Commons.
  3. Amino Acids forming a peptide bond (bottom image) by OpenLab at CitiTech CC-BY-NC-SA

Biomolecular Detection and Quantification

  • to provide a forum for discussion and recommendation of guidelines designed to improve the accuracy of molecular measurement, its data analysis and the transparency of its subsequent reporting
  • to publish molecular biology based studies that adhere to best practice guidelines, both current and future.
  • original research articles (both methodological and applied)
  • short communications
  • reviews
  • editorials and comments
  • points of view and perspective articles

All submitted manuscripts must present original contributions and contain no data that have been published elsewhere. Please note that BDQ operates with a so-called double blind peer-review policy which means that both the author and the reviewer remain anonymous to each other to avoid reviewer bias.

The journal has an international audience and we particularly welcome submissions from younger scholars and researchers and those from developing countries.

  • Genetics
  • Epigenetics
  • Transcriptomics
  • Proteomics
  • Metabolomics
  • Cancer
  • Infectious diseases/microbiology/virology
  • Diagnostics

Keywords: molecular quantification, molecular diagnostics, standardization, standardization, metrology, dpcr, qpcr, ngs, rnaseq, biomarkers, microbiome, metagenomics, epigenetics, infectious disease


Chromatography

Chromatography is the separation of sample components based on differential affinity for a mobile versus a stationary phase. The mobile phase is a liquid or a gas that flows over or through the stationary phase, which consists of spherical particles packed into a column. When a mixture of proteins is introduced into the mobile phase and allowed to migrate through the column, separation occurs because proteins that have a greater attraction for the solid phase migrate more slowly than do proteins that are more attracted to the mobile phase.

Several different types of interactions between the stationary phase and the substances being separated are possible. If the retarding force is ionic in character, the separation technique is called ion exchange. Proteins of different ionic charges can be separated in this way. If substances absorb onto the stationary phase, this technique is called absorption chromatography. In gel filtration or molecular sieve chromatography, molecules are separated because of their differences in size and shape. Affinity chromatography exploits a protein's unique biochemical properties rather than the small differences


Introduction

The term 𠇋iosensor” refers to powerful and innovative analytical device involving biological sensing element with wide range of applications, such as drug discovery, diagnosis, biomedicine, food safety and processing, environmental monitoring, defense, and security. The first biosensor invented by Clark and Lyons (1962) to measure glucose in biological samples utilized the strategy of electrochemical detection of oxygen or hydrogen peroxide (Fracchiolla et al., 2013 Turner, 2013) using immobilized glucose oxidase electrode. Since then, incredible progress has been made (Turner, 2013) both in technology and applications of biosensors with innovative approaches involving electrochemistry, nanotechnology to bioelectronics. Considering the phenomenal advances in the field of biosensors, this review is aimed to introduce various technical strategies, adopted for developing biosensors in order to provide fundamental knowledge and present scientific scenario of biosensor technology. With the emphasis on the research tools that demonstrate how the performance of biosensors evolved from the classical electrochemical to optical/visual, polymers, silica, glass, and nanomaterials to improve the detection limit, sensitivity, and selectivity. Interestingly, microbes and bioluminescence (Du et al., 2007) also contributed largely for label-based biosensors, while label-free biosensors involved usage of transistor or capacitor-based devices and nanomaterials. Biosensors provide a basis to understand technological improvement in the instrumentation involving sophisticated high-throughput machines for quantitative biologists and portable qualitative or semi-quantitative devices for non-specialists. Finally, current research trends, future challenges, and limitations in the field are highlighted. The present review is divided to various subsections describing two major technical strategies followed by various types of biosensor devices ranging from electrochemical, optical/visual, polymers, silica, glass, and nanomaterials. These devices were developed for specific purposes and an overview of these will provide readers a comprehensive data on biosensor devices and their applications.


Carbon Nanomaze for Biomolecular Detection with Zeptomolar Sensitivity

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Department of Clinical Laboratory Medicine, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038 China

Department of Clinical Laboratory Medicine, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038 China

Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Beijing, 400714 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Center of Smart Laboratory and Molecular Medicine, Medical School, Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Center of Smart Laboratory and Molecular Medicine, Medical School, Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Center of Smart Laboratory and Molecular Medicine, Medical School, Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Center of Smart Laboratory and Molecular Medicine, Medical School, Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Chongqing Key Laboratory of Bio-perception and Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Department of Clinical Laboratory Medicine, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038 China

Department of Clinical Laboratory Medicine, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038 China

Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Beijing, 400714 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Center of Smart Laboratory and Molecular Medicine, Medical School, Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Center of Smart Laboratory and Molecular Medicine, Medical School, Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Center of Smart Laboratory and Molecular Medicine, Medical School, Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Center of Smart Laboratory and Molecular Medicine, Medical School, Chongqing University, Chongqing, 400044 China

Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044 China

Chongqing Key Laboratory of Bio-perception and Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing, 400044 China

Abstract

Despite numerous efforts, the accurate determination of trace biomolecules with zeptomolar sensitivity remains elusive. Here, a 3D carbon nanomaze (CAM) electrode for the ultrasensitive detection of trace biomolecules such as nucleic acids, proteins, and extracellular vesicles is reported. The CAM electrode consists of an interlaced carbon fiber array on which intercrossed graphene sheets are vertically tethered in situ, permitting local confinement of trace molecules to increase molecular hybridization efficiency. Furthermore, a self-assembled DNA tetrahedron array adopts a rigid spatial conformation to guarantee the controllable arrangement of immobilized biological probes, facilitating analytical sensitivity and reproducibility. In a proof-of-concept experiment on detecting microRNA-155, a linearity of 0.1 aM to 100 nM and a sensitivity of 0.023 aM (23 zM) are achieved. With the optimal parameters, the proposed nanoelectrode demonstrates encouraging consistency with quantitative real-time polymerase chain reaction during clinical sample detection. Through simple functionalization by appending various biomolecular probes of interest, the developed CAM platform with ultrahigh sensitivity can be exploited as a versatile tool in environmental, chemistry, biology, and healthcare fields.


Fluorescence correlation spectroscopy for the detection and study of single molecules in biology

The recent development of single molecule detection techniques has opened new horizons for the study of individual macromolecules under physiological conditions. Conformational subpopulations, internal dynamics and activity of single biomolecules, parameters that have so far been hidden in large ensemble averages, are now being unveiled. Herein, we review a particular attractive solution-based single molecule technique, fluorescence correlation spectroscopy (FCS). This time-averaging fluctuation analysis which is usually performed in Confocal setups combines maximum sensitivity with high statistical confidence. FCS has proven to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solution, and in living cells. The introduction of dual-color cross-correlation and two-photon excitation in FCS experiments is currently increasing the number of promising applications of FCS to biological research. BioEssays 24:758–764, 2002. © 2002 Wiley Periodicals, Inc.


Abstract

Nanopores have become one of the most important tools for single-molecule sensing, but the challenge for selective detection of specific biomolecules still exists. In this contribution, we develop a new technique for sensing carcinoembryonic antigen (CEA), one of the important cancer biomarkers, using solid-state nanopores as a tool. The method is based on the specific affinity between aptamer (Apt) modified magnetic Fe3O4–Au nanoparticles (MNPs) and CEA, and the formed CEA–Apt–MNPs and remaining Apt–MNPs can transport the nanopores by applying a positive potential after magnetic separation. Due to the obvious particle size difference between CEA–Apt–MNPs and Apt-MPs, their corresponding blockage signals could be distinguished completely by the degree of the current decline. Moreover, the frequency of the blockage signals for CEA–Apt–MNPs is proportional to the concentration of CEA within certain limits, indicating that our designed nanopore sensing strategy can quantitatively detect CEA in complex samples. This work demonstrates that our designed nanopore-based strategy can be used for CEA sensing with good selectivity and sensitivity and also can be used to analyze other protein biomarkers for early diagnosis and monitoring of cancer, though the detection limit (0.6 ng/mL) is not relatively low. In future works, we plan to improve our detection limit by the improvement of the nanopipette preparation technology and detection method.


Biochemical analysis techniques

Biochemical analysis techniques refer to a set of methods, assays, and procedures that enable scientists to analyze the substances found in living organisms and the chemical reactions underlying life processes. The most sophisticated of these techniques are reserved for specialty research and diagnostic laboratories, although simplified sets of these techniques are used in such common events as testing for illegal drug abuse in competitive athletic events and monitoring of blood sugar by diabetic patients.

To perform a comprehensive biochemical analysis of a biomolecule in a biological process or system, the biochemist typically needs to design a strategy to detect that biomolecule, isolate it in pure form from among thousands of molecules that can be found in an extracts from a biological sample, characterize it, and analyze its function. An assay, the biochemical test that characterizes a molecule, whether quantitative or semi-quantitative, is important to determine the presence and quantity of a biomolecule at each step of the study. Detection assays may range from the simple type of assays provided by spectrophotometric measurements and gel staining to determine the concentration and purity of proteins and nucleic acids, to long and tedious bioassays that may take days to perform.

The description and characterization of the molecular components of the cell succeeded in successive stages, each one related to the introduction of new technical tools adapted to the particular properties of the studied molecules. The first studied biomolecules were the small building blocks of larger and more complex macromolecules, the amino acids of proteins, the bases of nucleic acids and sugar monomers of complex carbohydrates. The molecular characterization of these elementary components was carried out thanks to techniques used in organic chemistry and developed as early as the nineteenth century. Analysis and characterization of complex macromolecules proved more difficult, and the fundamental techniques in protein and nucleic acid and protein purification and sequencing were only established in the last four decades.

Most biomolecules occur in minute amounts in the cell, and their detection and analysis require the biochemist to first assume the major task of purifying them from any contamination . Purification procedures published in the specialist literature are almost as diverse as the diversity of biomolecules and are usually written in sufficient details that they can be reproduced in different laboratory with similar results. These procedures and protocols, which are reminiscent of recipes in cookbooks have had major influence on the progress of biomedical sciences and were very highly rated in scientific literature.

The methods available for purification of biomolecules range from simple precipitation, centrifugation, and gel electrophoresis to sophisticated chromatographic and affinity techniques that are constantly undergoing development and improvement. These diverse but interrelated methods are based on such properties as size and shape, net charge and bioproperties of the biomolecules studied.

Centrifugation procedures impose, through rapid spinning, high centrifugal forces on biomolecules in solution, and cause their separations based on differences in weight. Electrophoresis techniques take advantage of both the size and charge of biomolecules and refer to the process where biomolecules are separated because they adopt different rates of migration toward positively (anode) or negatively (cathode) charged poles of an electric field. Gel electrophoresis methods are important steps in many separation and analysis techniques in the studies of DNA , proteins and lipids. Both western blotting techniques for the assay of proteins and southern and northern analysis of DNA rely on gel electrophoresis. The completion of DNA sequencing at the different human genome centers is also dependent on gel electrophoresis. A powerful modification of gel electrophoresis called twodimensional gel electrophoresis is predicted to play a very important role in the accomplishment of the proteome projects that have started in many laboratories.

Chromatography techniques are sensitive and effective in separating and concentrating minute components of a mixture and are widely used for quantitative and qualitative analysis in medicine, industrial processes, and other fields. The method consists of allowing a liquid or gaseous solution of the test mixture to flow through a tube or column packed with a finely divided solid material that may be coated with an active chemical group or an adsorbent liquid. The different components of the mixture separate because they travel through the tube at different rates, depending on the interactions with the porous stationary material. Various chromatographic separation strategies could be designed by modifying the chemical components and shape of the solid adsorbent material. Some chromatographic columns used in gel chromatography are packed with porous stationary material, such that the small molecules flowing through the column diffuse into the matrix and will be delayed, whereas larger molecules flow through the column more quickly. Along with ultracentrifugation and gel electrophoresis, this is one of the methods used to determine the molecular weight of biomolecules. If the stationary material is charged, the chromatography column will allow separation of biomolecules according to their charge, a process known as ion exchange chromatography. This process provides the highest resolution in the purification of native biomolecules and is valuable when both the purity and the activity of a molecule are of importance, as is the case in the preparation of all enzymes used in molecular biology . The biological activity of biomolecules has itself been exploited to design a powerful separation method known as affinity chromatography. Most biomolecules of interest bind specifically and tightly to natural biological partners called ligands: enzymes bind substrates and cofactors, hormones bind receptors, and specific immunoglobulins called antibodies can be made by the immune system that would in principle interact with any possible chemical component large enough to have a specific conformation. The solid material in an affinity chromatography column is coated with the ligand and only the biomolecule that specifically interact with this ligand will be retained while the rest of a mixture is washed away by excess solvent running through the column.

Once a pure biomolecule is obtained, it may be employed for a specific purpose such as an enzymatic reaction, used as a therapeutic agent, or in an industrial process. However, it is normal in a research laboratory that the biomolecule isolated is novel, isolated for the first time and, therefore, warrants full characterization in terms of structure and function. This is the most difficult part in a biochemical analysis of a novel biomolecule or a biochemical process, usually takes years to accomplish, and involves the collaboration of many research laboratories from different parts of the world.

Recent progress in biochemical analysis techniques has been dependant upon contributions from both chemistry and biology, especially molecular genetics and molecular biology, as well as engineering and information technology. Tagging of proteins and nucleic acids with chemicals, especially fluorescent dyes , has been crucial in helping to accomplish the sequencing of the human genome and other organisms, as well as the analysis of proteins by chromatography and mass spectrometry. Biochemical research is undergoing a change in paradigm from analysis of the role of one or a few molecules at a time, to an approach aiming at the characterization and functional studies of many or even all biomolecules constituting a cell and eventually organs. One of the major challenges of the post-genome era is to assign functions to all of the gene products discovered through the genome and cDNA sequencing efforts. The need for functional analysis of proteins has become especially eminent, and this has led to the renovated interest and major technical improvements in some protein separation and analysis techniques. Two-dimensional gel electrophoresis, high performance liquid and capillary chromatography as well as mass spectrometry are proving very effective in separation and analysis of abundant change in highly expressed proteins. The newly developed hardware and software, and the use of automated systems that allow analysis of a huge number of samples simultaneously, is making it possible to analyze a large number of proteins in a shorter time and with higher accuracy. These approaches are making it possible to study global protein expression in cells and tissues, and will allow comparison of protein products from cells under varying conditions like differentiation and activation by various stimuli such as stress, hormones, or drugs. A more specific assay to analyze protein function in vivo is to use expression systems designed to detect protein-protein and DNA-protein interactions such as the yeast and bacterial hybrid systems. Ligand-receptor interactions are also being studied by novel techniques using biosensors that are much faster than the conventional immunochemical and colorimetric analyzes.

The combination of large scale and automated analysis techniques, bioinformatic tools, and the power of genetic manipulations will enable scientists to eventually analyze processes of cell function to all depths.

See also Bioinformatics and computational biology Biotechnology Fluorescence in situ hybridization Immunological analysis techniques Luminescent bacteria


Chapter Four - Enzymes as Sensors

Over the last few decades the development of new technologies, the fabrication of new materials, and the introduction of nanotechnologies created new trends in a series of advances that produced innovations in biological sensing devices with a wide range of application from health, security, defense, food, and medicine, to the environment. Specificity, low cost, rapidity, sensitivity, and multiplicity are some of the reasons for their growth, and their commercial success is expected to increase in the next future.

Biosensors are devices in which the recognition part of the target molecule is accomplished by biological macromolecules such as proteins, enzymes, antibodies, aptamers, etc. These biomolecules are able to bind to the target molecules with high selectivity and specificity. The interaction between the target molecule and the specific biomolecule is reflected as a change of the biomolecule structural features. The extent of this change is strictly related to the biosensor response. Fluorescence spectroscopy, due to its sensitivity, is often used as the principal technique to monitor biological interactions, and thus the biosensor response as well. Both the intrinsic ultraviolet fluorescence of protein, arising from aromatic amino acids (tryptophan, tyrosine, and phenylalanine), and extrinsic fluorescent labels emitting in the visible region of the spectrum together allow for very flexible transduction of the analyte recognition, suitable for many different applications.

This chapter focuses special attention on enzymes as practically unmatched recognition elements for biosensors and emphasizes the potential advantages of customized biosensor devices using apo- or holo forms of enzymes also isolated from thermophile sources.


Chip for biomolecule detection may help in COVID-19 testing

A patented method for single biomolecule detection that overcomes limitations of current technologies may help in the fight against COVID-19.

Purdue University innovators created a method that uses a special sensor similar to a computer chip. The application-specific integrated circuit chip is designed for the early detection of a number of pathogens and viruses.

“We want to find partners to move this technology to the public as soon as we can to help in COVID-19 testing,” said Saeed Mohammadi, a Purdue professor of electrical and computer engineering. “We know it can be an effective, easy and inexpensive method for detecting viruses, potentially the one linked to the current pandemic.”

The Purdue technique involves machine learning to train the system to detect certain features associated with particular diseases and viruses. Then, when a sample is run through the system, it can detect those features and confirm the presence of particular viruses and diseases. Simulations have shown this technique may be effective in detecting COVID-19.

This method uses a metal-oxide semiconductor sensor with embedded, fluidic nanochannels. As a biomolecule moves through the nanochannel, a high frequency current is measured that contains information about the biomolecule, such as the type of nucleotides in the case of DNA/RNA, which can be used to classify the molecule.

Mohammadi said, “This method does not have the problems associated with other nanopore techniques because it does not require the difficult drilling of extremely small nanopores, can detect four nucleotides at a time, and is not significantly affected by the rotation or position of the biomolecule in the nanochannel.”

Mohammadi said the technology is simple enough that a manufacturer could use it to develop a test kit that could be used at home for virus and disease detection.

The team worked with the Purdue Research Foundation Office of Technology Commercialization to patent this technology. The office recently moved into the Convergence Center for Innovation and Collaboration in Discovery Park District, adjacent to the Purdue campus.

The researchers are looking for partners to continue developing their technology. For more information on licensing and other opportunities, contact Matthew Halladay of OTC at [email protected] and mention track code 2013-MOHA-66407.


Watch the video: Biological Molecules Part I (May 2022).


Comments:

  1. Hassan

    In this the whole thing.

  2. Roslin

    It is very curious:)

  3. Ra

    How can I contact you, the fact is that I have been developing this topic for a long time and it is very pleasant to find like-minded people.

  4. Sherbourn

    Of course. I agree with all of the above. We can communicate on this theme. Here or at PM.

  5. Dary

    This is not always the case.



Write a message