BIOSENSORS, THEIR TYPES AND THEIR APPLICATIONS IN THE MOLECULAR IMAGING OF HYPOXIA IN TUMORS

A Mid-Term Status Update

Presented By Supervised By D Devansh Dr. Ritu Kulshreshtha(2009BB50009)

Department of Biochemical Engineering and Biotechnology IIT Delhi, New Delhi-110016

OBJECTIVE :

The aim of this study is understanding Biosensors, along with their various types and put this knowledge to use in understanding the applications of Biosensors in the molecular imaging of Hypoxic Tumors.

OVERVIEW OF THE PLAN OF ACTION: Understanding the working of Biosensors. Sorting different kinds of Biosensors and analyzing their Pros and Cons. Identifying the areas in which Biosensors have applications. Understanding of Tumor Hypoxia and its consequences. Extensive Study of current methods of detection of hypoxic tumors. Using the mined-out knowledge to try and introduce a novel aspect to existing

methods.

Biosensors - An Introduction

Biosensors are tools that can biologically react to an analyte and transmit a measurable signal representing the interaction.

Typical Characteristics of Biosensors include:• High Specificity

• Lock and Key Mechanism, Ab-Ag interaction etc.• High Sensitivity

• High affinity of biomolecules.• Easy to Manufacture

• Advances in Electronics and also in synthetic construction of aptamers.

WHAT ARE BIOSENSORS?

Most biosensors are composed of three components:

1. Bio-Receptor2. Transducer3. Signal Processor

BIOSENSOR-COMPONENTS

• Enzyme-based Biosensors

• Immunological Biosensors

• Nucleic Acid Biosensors

TYPES OF BIOSENSORS

Employ Enzyme-Substrate binding as bio-recognition part of the biosensor.

Typically, the enzyme is immobilized at an electrode, where it binds solely to its substrate.

An example is the blood glucose detector:

ENZYME-BASED BIOSENSORS

In a blood glucose detector the enzyme glucose oxidase is immobilized at an electrode and binds to glucose in presence of oxygen to give gluconic acid.

The detection is done by measuring the concentration of oxygen through an oxygen electrode and then calibrating it to estimate the glucose concentration.

Downsides include: Difficulty of enzyme extraction Instability of some enzymes Co-factors are needed for some enzymes

As the name suggests, these biosensors depend on the Antibody-Antigen binding.

Either the Antibody or the Antigen can be immobilized at an electrode, which would bind to the corresponding target.

ELISA (Enzyme linked Immunosorbent Assay) is a common immunological biosensor. A sandwich ELISA is described next.

IMMUNOLOGICAL BIOSENSOR

NUCLEIC ACID BIOSENSORS (Genosensors)

These generally utilize the complementarity of DNA/RNA binding.

These can be of two types Interaction between complementary single stranded nucleic acid

elements (Aptamers) Interaction between a Nucleic Acid sequence with a particular protein.

( TF binding to Promoter elements)

Most “aptamers” are not more than 100 nucleotides long.

Genosensors, can be further divided into RNA and DNA.

mRNA molecules, unlike DNA, are degraded rapidly in living bacterial cells because of the presence of RNAases.

This make RNA favorable to detect viable cells. (finds applications in food industry)

In other cases DNA may be favored because of higher stability.

Name of the bio-receptor.

Size Synthetic Ease of Production

Durability Binding Affinity Specificity

DNA Small Easy High High Highly specific

RNA Small Easy(one more step than DNA if 2’

pyrimidine position is to be modified)

Moderate (susceptible to

attack on2’ pyrimidine if not modified)

High Highly Specific

Antibodies, Enzymes

Large Hard( because of large) and extraction is also

difficult.

Moderate(found to be less

durable than RNA and DNA)

High Moderately Specific

COMPARING BIOSENSOR TYPES

In Health Care:

Biosensors are needed in health care to detect analytes which are used to determine the stability of the patient’s metabolic activities.

One of the most popular application here has been measurement of blood glucose concentration in diabetes patients.

AccuChek™ is one of the major corporations that makes use of this technology commercially.

• BIOSENSOR: APPLICATIONS

In Process Control:

Biosensors can potentially be used to monitor carbon sources, dissolved gases and other process parameters.

Currently, only a few process control parameters have real time monitoring like pH, DO and temperature.

It has been suggested that use of biosensors in measuring other parameters (carbon source, gases etc)could better facilitate plant automation, cut analysis costs and also improve quality control of the product.

Hypoxia literally means “low oxygen”. When it occurs in Tumor cells it is called Tumor Hypoxia.

Caused due to excessive cell growth in aggressive tumors.

Grants Chemotherapeutic resistance to tumors.

Hence, Poorer prognosis.

Therefore, it is important to detect these kind of tumors.

• HYPOXIA IN TUMORS: INTRO

HIF-1: Hypoxia inducible factor(HIF-1α + HIF-1β )

Induced when oxygen levels are low, else degraded.

Activates various other proteins like VEGF and GLUT-1.

Also can cause p53 mutation, leading to cell immortality.(more aggressive tumor)

VEGF mediated Angiogenesis, which brings in essential nutrients to help the tumor survive.

• HYPOXIC TUMORS: PROBLEMS

• Hypoxia in tumors is found to grant them resistance to therapy.

Many drugs require the presence of cellular oxygen to function.

p53 mutations lead to loss of apoptotic ability.

Abnormal Vasculature causes reduced drug diffusion.

• CHEMOTHERAPEUTIC RESISTANCE

Using positron emission tomography (PET) and a hypoxia marker, tumor hypoxia can be detected in gliomas

Different techniques have been developed depending on the hypoxia marker used.

Some markers are – (FMISO,FRP-170 and FAZA (based on isotopes of fluorine) and also IAZA( iodine based). All are derivatives of 2- nitroimidazole.

Nucleic acid based sensor exploiting the HRE binding of HIF-1 to detect hypoxia.

• SOME METHODS OF IMAGING HYPOXIC TUMORS

The Method:• Bio-Receptor: An E-M-H enhancer. ACGTGG is the recognition sequence on

most HREs.

• Transducer: A Fusion Protein (mCherry protein+ Luciferase)

• EMH enhancer is activated by HIF-1( Transcription Factor). Leading to expression of the fusion protein.

• A BIOSENSOR FOR HYPOXIC TUMOR DETECTION

Plasmid used as biosensor [31]

Gene HRE core sequence

Eno-1, human GGGCCGGACGTGGGGCCCCAGAGCGA

Epo, human GGGCCCTACGTGCTGTCTCACACAGC

Epo, murine GGGCCCTACGTGCTGCCTCGCATGGC

GLUT-1, murine TCCACAGGCGTGCCGTCTGACACGCA

iNOS, human TGACTACGTGCTGCCTAG

LDH-A, murine CCAGCGGACGTGCGGGAACCCACGTG

PFK-L, murine CTGGCGTACGTGCTGCAG

PGK-1, human GTGAGACGTGCGGCTTCC

PGK-1, murine TTGTCACGTCCTGCACGA

VEGF, human AGTGCATACGTGGGCTCCAACAGGTC

VEGF, murine AGTGCATACGTGGGTTTCCACAGGTC

VEGF, rodent AGTGCATACGTGGGCTTCCACAGGTC

Work to be done includes reading more about other ways of hypoxia detection.

After this, Analyzing each studied method and their pros and cons.

If possible, introducing a novel aspect to the present literature.

•WORK TO BE DONE:

• 1) Benson DE, Conrad DW, de Lorimier RM, et al. Design of bioelectronic interfaces by exploiting hinge-bending motions in

proteins. Science. 2001;293:1641–4.

• 2) Boon EM, Ceres DM, Drummond TG, et al. Mutation detection by electrocatalysis at DNA-modified electrodes. Nature

Biotech. 2000a;18:1096–100.

• 3) Boon EM, Livingston AL, Chmiel NH, et al. DNA-mediated charge transport for DNA repair. Proc Natl Acad Sci U S A.

2003;100:12543–7.

• 4) Boon EM, Salas JE, Barton JK. An electrical probe of protein-DNA interactions on DNA-modified surfaces . Nat Biotechnol.

2002;20:282–6.

• 5) Bowtell DDL. Options available – from start to finish – for obtaining expression data by microarray . Nat Genet. 1999;21:25–

32.

• 6) Brazill SA, Kim PH, Kuhr WG. Capillary gel electrophoresis with sinusoidal voltammetric detection: A strategy to allow

four-“color” DNA sequencing. Anal Chem. 2001;73:4882–90.

• 7) Burgstaller P, Girod A, Blind M. Aptamers as tools for target prioritization and lead identification. Drug Discov Today.

2002;7:1221–8.

• 8) Chang KY, Varani G. Nucleic acids structure and recognition. Nature Struct Biol. 1997;4:854–8.

REFERENCES USED

• 9) Clark JLC, Lyons C. Electrode systems for continuous monitoring in cardiovascular surgery. Ann N Y Acad Sci. 1962;102:29–

45.

• 10) Cooper MA, Dultsev FN, Minson T, et al. Direct and sensitive detection of a human virus by rupture event scanning. Nature

Biotechnol. 2001;19:833–7.

• 11) Dai Z, Yan F, Yu H, et al. Novel amperometric immunosensor for rapid separation-free immunoassay of carcinoembryonic

antigen. J Immuno Methods. 2004;287:13–20.

• 12) Drummond TG, Hill MG, Barton JK. Electrochemical DNA sensors. Nat Biotechnol. 2003;21:1192–9.

• 13) Fan C, Plaxco KW, Heeger AJ. Electrochemical interrogation of conformational changes as a reagentless method for the

sequence-specific detection of picomolar DNA. Proc Natl Acad Sci U S A. 2003;100:9134.

• 14) Fan C, Plaxco KW, Heeger AJ. Biosensors based on binding-modulated donor-acceptor distances. Trends Biotechnol.

2005;23:186–92.

• 15) Fritz J, Cooper EB, Gaudet S, et al. Electronic detection of DNA by its intrinsic molecular charge. Proc Natl Acad Sci U S A.

2002;99:14142–6.

• 16) Gao Z, Binyamin G, Kim H-H, et al. Electrodeposition of redox polymers and co-electrodeposition of enzymes by

coordinative crosslinking. Angew Chem Int Ed. 2002;41:810–13.

• 17) Gaylord BS, Heeger AJ, Bazan GC. DNA detection using water-soluble conjugated polymers and peptide nucleic acid

probes. Proc Nat Acad Sci U S A. 2002;99:10954.

• 21) Ihara T, Maruo Y, Takenaka S, et al. Ferrocene-oligonucleotide conjugates for electrochemical probing of DNA. Nucleic Acids

Res. 1996; 24:4273–80.

• 22) Kelley SO, Barton JK. Electron transfer between bases in double helical DNA. Science. 1999; 283:375–81.

• 23) Kelley SO, Jackson NM, Hill MG, et al. Long-range electron transfer through DNA films. Angew Chem Int Ed. 1999; 38:941–5.

• 24) Kuhr WG. Electrochemical DNA analysis comes of age. Nature Biotech. 2000; 18:1042–3.

• 25) Millan KM, Mikkelsen SR. Sequence-selective biosensor for DNA-based on electroactive hybridization indicators. Anal Chem.

1993; 65:2317–23.

• 26) Mrksich M, Whitesides GM. Using self-assembled monolayers to understand the interactions of man-made surfaces with

proteins and cells. Annu Rev Biophys Biochem. 1996; 25:55–78.

• 27) Palecek E. Surface-attached molecular beacons light the way for DNA sequencing. Trends Biotechnol. 2004; 22:55–8.

• 28) Palecek E, Jelen F. Electrochemistry of nucleic acids and development of DNA sensors. Crit Rev Anal Chem. 2002; 32:261–70.

• 29) Song S, Xu H, Fan C. Potential diagnostic applications of biosensors: current and future directions . Int J Nanomedicine.

2006;1(4):433–40

• 30) doi:10.1016/S0305-7372(03)00003-3.

• 31) doi:10.1016/j.bios.2009.04.013

• 32) doi :10.3390/s8074296