Biomedical Engineering Trends Research and Technologies Part 12 pptx

19 The Use of Phages and Aptamers as Alternatives to Antibodies in Medical and Food Diagnostics Jaytry Mehta1,2, Bieke Van Dorst1,2, Lisa Devriese2 Elsa Rouah-Martin1,2, Karen Bekaert

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J (2010) The influence of biocomposites containing genetically modified flax fibers

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18

Characterization of Hydroxyapatite Blocks

for Biomedical Applications

Masoume Haghbin Nazarpak1, Mehran Solati-Hashjin2 and Fatollah Moztarzadeh2

1New Technologies Research Center, Amirkabir University of Technology,

2Department of Biomedical Engineering, Amirkabir University of Technology,

Iran

1 Introduction

There is an increasing demand for materials to be used in biomedical and dental applications These materials are currently implemented in different forms, depending on the part of the body which needs repair Biocompatibility, biofunctionality, and availability are three significant factors in selecting materials (L.L Hench, 1998)

Historically, ceramics are the oldest materials in medical applications Tricalcium phosphate (TCP) was used for repairing bone defects in the early 20th century (M Bohner, 2000; T Cuneyt, et al 1997; J.G.J Peelen, et al 1987) Although, ceramics are brittle by nature, they have excellent compressive strength and a high wear resistance Calcium phosphate compounds such as hydroxyapatite (HA), tricalcium phosphate (TCP), dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous (DCPA), and tetracalcium phosphate (TTCP) (L.L Hench, 1998; M Bohner, 2000; T Cuneyt, et al 1997; J.G.J Peelen, et al 1987; C Laverinia & J.M Schoenung, 1991; M Komath, et al 2000; S.Takagi, et al 1998) have almost the same chemical compositions as bone minerals When these compounds are implanted

into the living body (in vivo) for a period of time, they create a strong chemical bond with

bone tissue (P Luo, et al 1998; H.H Pham, et al 1999)

In replacing bone defects, besides all compatibility parameters, the material should possess the same porosity as the bone Bone has a complex structure with macro- and micro-pores Pores are mostly interconnected to allow body fluids to carry nutrients and provide a medium where interfacial reactions between hard tissue and soft tissue can occur An implant material should generally present similar properties to that of the bone However, mechanical requirements dictate a high strength for implants which is associated with the elimination of some pores from them As a result, reducing the porosity should result in an increase on the mechanical properties of HA as with any other ceramics It is therefore important to find an optimum porosity to maintain the mechanical strength while pores provide the bone implant with an acceptable channel for nutrition to obtain the best implant properties (M Jarcho, 1981; J.C Le Hiec, et al 1995)

The aim of the present work was to find the effect of sintering temperature on the microstructure, phase composition and the mechanical properties of hydroxyapatite ceramics

2 Materials and methods

Medical grade hydroxyapatite powder was obtained from Sigma-Aldrich Chemical Company The density of the powder was measured as 2.91 g/cm3 using ACCU-PYC 1330 (Micromeritics Gemini 2375) In order to determine the sintering temperature, a dilatometry test was performed Also, the X-ray diffraction technique was employed using a Siemens,

D500 diffractometer at each sintering temperature to estimate the probable phase

transformations and the upper limit of the decomposition temperature The particle size

distribution was measured using a Fritsch Analysette22 system The starting powder was

uniaxially compacted at 86 MPa to form cylindrical shaped samples 55* 13 mm The sintering was performed in air at 700-1300 °C with 1 hour soaking time The rate of temperature increase was 10 °Kh-1 while the cooling was carried out in the furnace The mechanical properties of sintered bodies were examined by 3 and 4 point bending techniques using an Instron Universal Testing Machine 1196 was used with a cross head speed of 0.5 mm/min and maximum load application of 5 kN Vickers hardness of specimens was measured using Vickers hardness testing device (Hardness Tester Akashi AVK cll) under 300 g loading at 20 seconds Fracture toughness also was determined using Evans & Charles equation The microstructures of the samples were studied under different sintering conditions using a Cambridge Stereosacn 360 scanning electron microscope

3 Results and discussion

Figure 1 shows the particle size distribution It is evident that the average size of the powder was around 4 micrometre The particle size and specific surface area of the starting powder are the most important parameters affecting the sintering behavior of ceramics By reducing the particle size and increasing the specific surface area, the same degree of sintering can be achieved at much lower temperatures

Fig 1 Particle size distribution of the hydroxyapatite powder

The specific surface area of the starting powder was measured as 52.3 m2/g which compared with other commercial powders can be considered as an active powder The

Characterization of Hydroxyapatite Blocks for Biomedical Applications 437 molar ratio of Ca/P is another important parameter which is 1.5 in tricalcium phosphate, 2

in tetra calcium phosphate, and 1.67 in stoichiometric hydroxyapatite (A Siddharthan, et al 2005) The molar ratio of the Ca/P in the present powder was measured with the ICP technique at around 1.62 A ratio less than 1.64 will be interpreted as the creation of pores and voids in a sintered body A ratio higher than 1.67 means that the rate of absorption in vivo will be increased (P Vincenzini, 1986)

In order to gain insights into the sintering behavior of hydroxyapatite, dilatometric tests were carried out Figure 2 shows a typical curve which indicates the shrinkage starts at about 700 °C and the sintering temperature is estimated to be between 900 and 1300 °C (K.A Hing, et al 2000; A.J Ruys, et al 1995; B.J Meenan, et al 2000; L.L Hench, 1991; M Jarcho,

et al 1976; M.K Sinha, et al 2000; A.J Ruys, et al 1995; G De With, et al 1981; P Landuyt,

et al 1995; M.Y Shareef et al 1993; J Zhou, et al 1993)

01234

Fig 2 Dilatometry measurement for a hydroxyapatite sample

XRD results of the sintered samples are shown in figure 3a to 3d As it is evident from these patterns, within the sintering range of 700-1300 °C, no other phases could be detected by XRD and the results confirmed formation of hydroxyapatite phase Also, the CaO phase was checked for in particular to be absent since this phase has been shown to have negative effects on the growth of bone cells (K.A Hing, et al 2000)

The Vickers hardness of specimens was measured through indentation method The results showed rising hardness from 1.5 to 6.1 GPa with increasing sintering temperature from 1100

° C to 1300 ° C that is shown in Figure 4 Fracture toughness of samples was also calculated from Evans & Charles equation was approximately between 0.5 and 0.85 MPam1/2 at sintering temperature about 1200° C

In order to make microscopic studies, samples were etched in 1% phosphoric acid and gold sputtered prior to study by SEM studies Figure 5 shows scanning electron microscope images of hydroxyapatite sintered at different temperatures In all cases, the sintered samples were highly polished with different grades of polish; the last one being 1 micrometre of diamond paste

Fig 3 X-ray diffraction pattern of: a) starting powder, b) fired at 900 °C for 1 hour, c) fired at

1100 °C for 1 hour, d) fired at 1300 °C for 1 hour

Characterization of Hydroxyapatite Blocks for Biomedical Applications 439

01234567

As is evident from figure 5(a), when the samples were fired at 1100 °C, hardly any changes

in grain size could be observed Sintering at 1200 °C results in grain growth in the microstructure of samples as is shown in figure 5(b) The grain growth is also associated with a reduction in the apparent porosity which is a favorable condition as far as the mechanical strength of the part is concerned

Figure 5(c) shows an electron micrograph of a sample sintered at 1300 °C Further grain growth is evident and a higher mechanical strength is expected for this sample which is in accordance with bending strength results

The mechanical properties of sintered bodies were examined by 3 and 4 point bending techniques after samples polished with emery paper and diamond paste An Instron Universal Testing Machine 1196 was used with a cross head speed of 0.5 mm/minute and maximum load application of 5 kN In fact, in many cases, the mechanical properties of the samples were improved when they were fired at higher temperatures as is demonstrated in figures 6 a and b

Fig 6 Bending strength against sintering temperature a) 3 point and b) 4 point

Characterization of Hydroxyapatite Blocks for Biomedical Applications 441

4 Conclusions

The present study revealed that the sintering of hydroxyapatite at 1100- 1300 °C results in an essentially porous body which can be used as an implant XRD diffraction patterns of the sintered ceramics proved that no additional phase formation takes place even at elevated temperatures

Mechanical measurements proved that the bending strength of the sintered bodies were between 7-44 MPa which improved proportional with sintering temperature Microstructural studies showed that while the grain size of the bodies sintered at 1100 °C remained basically comparable to the particle size of the starting powder, ceramics fired at

1200 to 1300 °C showed an increase in grain size in line with the increased temperature

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Part 6 Advances in Diagnostics

19

The Use of Phages and Aptamers as Alternatives to Antibodies in Medical and

Food Diagnostics

Jaytry Mehta1,2, Bieke Van Dorst1,2, Lisa Devriese2

Elsa Rouah-Martin1,2, Karen Bekaert2, Klaartje Somers3,

Veerle Somers3, Marie-Louise Scippo4, Ronny Blust1 and Johan Robbens1,2

1University of Antwerp, Department of Biology, Laboratory of Ecophysiology, Biochemistry and Toxicology, Groenenborgerlaan 171, 2020 Antwerp

2Institute for Agricultural and Fisheries research (ILVO), Ankerstraat 1, 8400 Oostende

3Hasselt University, Biomedical Research Institute, B-3590 Diepenbeek

4University of Liège, Food Sciences Department, B-4000 Liège

in the medical field for rapid, cheap and reliable diagnostic systems in order to detect all the well-known and recently identified biomarkers for different diseases This identification is not a trivial exercise because these disease biomarkers are present in minute quantities in physiological conditions such as the bloodstream or body fluids, which are often contaminated with many other compounds that can hinder detection Apart from biomarker diagnosis, another area of concern for human health has been food contamination Trading

of contaminated food between countries and high population mobility increases the potential for outbreaks and health risks posed by microbial pathogens and toxins in food Food safety has become a global health goal Periodic toxin and microbiological analyses of food samples are important to diagnose and prevent problems related to health and food safety However, food-borne pathogens are mostly present in very low numbers among various other microorganisms, making their detection difficult To be able to detect these disease carriers and biomarkers in their natural conditions, highly sensitive as well as specific recognition elements are required It is necessary to develop detection techniques that are reliable, fast, easy, sensitive, selective, cost-effective and also suitable for real time,

in situ monitoring Such techniques to detect pathogens and biomarkers would not only

improve clinical success rates but also offer a great commercial advantage to the medical field and the food industry

Conventional techniques for the detection of disease biomarkers, pathogens and toxins are immunology-based methods, polymerase chain reaction (PCR) based methods and culture and colony counting methods Though these standard detection methods are sensitive, they lag behind in terms of detection time, taking from several hours to days to yield a response (Velusamy et al 2010) Conventional analytical techniques like optical, chromatographic and electrochemical detection are faster, but have some limitations of equipment and cost Furthermore, they are complicated and require highly trained personnel and extensive

sample preparation These constraints do not always allow frequent, real time or in situ

monitoring of food or clinical samples Thus, demands of high sensitivity, specificity, effective, portable and rapid analyses have propelled the development of biosensors as novel diagnostic tools in the medical and food sectors

cost-Many diagnostic tools still rely on immunoassays and especially enzyme-linked immunosorbent assay (ELISA) Besides these classical tests, several phage and aptamer based sensors have also been proposed for a broad range of disease biomarkers or carriers such as antibodies, viruses, disease-related proteins, tumour cells, toxins and pathogens, among several others The precise detection of these biomarkers or carriers before the onset

of a disease can significantly revolutionise the medical field by providing cheap, fast, simple and easily produced diagnostic tests using phages and aptamers as their recognition elements The wide range of assays that employ phages or aptamers to detect important clinical molecules, highlights the potential of these new receptors in clinical diagnostic tests and in food biosensors

This chapter will define phages and aptamers and discuss their use as novel biorecognition elements in biosensors We discuss two relevant cases in the field of biosensors: the use of diagnostics for clinical testing and the use of biosensors for food-related testing

2 Biosensors

A biosensor is defined by the IUPAC as a self-contained integrated device that is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor), which is retained in direct spatial contact with a transduction element

Biosensors can be categorised by the type of recognition element used, such as enzymatic, whole cell or affinity-based biosensors Enzymes are proteins that catalyse specific chemical reactions and were the first molecular recognition elements to be included in biosensors They are attractive sensor recognition elements because their use can convert the analyte into a sensor-detectable product, evaluate the modification of enzyme properties upon interaction with the analyte or detect an analyte that acts as the enzyme inhibitor or activator In fact, the most widely studied and acclaimed sensor success story is that of the glucose biosensor, an enzymatic sensor (Newman & Turner 2005; Wang 2007) Whole-cell biosensors, the next classification of biosensors, often use a genetically engineered cell of either eukaryotic or prokaryotic origin, containing responsive transcriptional promoter elements as the biological component These whole cell biosensors are used for the profiling

of the toxicological effects of compounds and for risk assessment of chemical contaminants

or of new compounds (Robbens et al 2010) The third type of biosensors which are of higher relevance for diagnostics, are affinity-based biosensors In these, the affinity-based receptor

The Use of Phages and Aptamers as Alternatives to Antibodies in Medical and Food Diagnostics 447 molecule binds the analyte irreversibly and non-catalytically The binding event between the target molecule and the bioreceptor, triggers a physicochemical change that can be measured by a transducer In order to ‘visualise’ the binding event, the different transduction methods that are frequently used in these biosensors are optical, electrochemical or mass-based

When the detection system requires a biomolecular recognition event, antibody-based detection methodologies are considered the standard assays in clinical analysis (Aizawa 1994; Stefan et al 2000) These assays are well established and have been demonstrated to

reach the required sensitivity and selectivity However, the use of antibodies in situ

detection methods and in the analysis of very complex samples could encounter some limitations mainly deriving from the nature and synthesis of these protein receptors Antibodies are relatively cheap, but their production relies on the immune response of an animal Besides the ethical problems related to the use of animals, it is also difficult to generate antibodies for toxic compounds or small compounds that cannot elicit an immune response In order to avoid some of these drawbacks, recent advances in biotechnology, nanotechnology and surface chemistry offer the possibility of developing other novel, affinity-based recognition molecules that have been explored as alternatives to the traditionally used antibodies This is a domain in which phages and aptamers can play a successful role They have emerged as viable options thanks to their high selectivity and affinity towards their targets, comparable to that of antibodies The high affinity and hence high sensitivity, high specificity, robustness, animal-friendly production and ease of modification are some of the defining properties that make the use of aptamers and phages advantageous in diagnostic and biosensing tools (Van Dorst et al 2010b)

3 Phages

Phages are viruses that use their host bacterial cells as factories for their own replication and have the ability to display peptides or proteins on their surfaces This technology is called phage display Phage display can be used as a powerful tool to screen for affinity reagents for all kind of targets, ranging from small molecules to proteins and even cells This selection can be performed by using phage libraries consisting of a high number of different phages (108 - 1010), each displaying a different peptide or protein on its surface Among the huge number of phages in these phage libraries, the ones with high affinity and specificity for a target can be isolated in an affinity selection procedure (Fig 1) Moreover, the proteins and peptides displayed on these selected phages can be identified by sequencing the gene coding for the displayed protein or peptide This coding gene can be found in the single-stranded DNA (ssDNA) inside the selected phage The target specific phages can be used as affinity reagents in diagnostic tests Besides the target specific phages, the soluble peptides

or proteins, released from the phage coat, can also be used as affinity reagents These peptides or proteins are then produced synthetically or by recombinant expression in bacterial cells

Different types of phage libraries exist, displaying different types of peptides or proteins: peptides, cellular proteins (from cDNA libraries) or antibody fragments, like single chain

variable fragments (scFv) and antigen binding fragment (Fab) Antibody fragment phage libraries are used commonly in immunology (Hoogenboom et al 1998) Their wide

diversity enables them to imitate the natural immune system Phage display enables the production of sizeable amounts of the affinity reagents, avoiding the batch to batch

Fig 1 Schematic representation of the phage affinity selection procedure (Van Dorst et al 2010a)

variations which occur with classical antibodies Moreover, there is no immune response required to produce affinity reagents for a target, making the selection of affinity reagents

for poorly immunogenic targets possible Peptide phage libraries can also be used to select

affinity reagents, since the binding site, or epitope, only involves a few amino acids Besides the monovalent phages that display one peptide on the phage surface, so-called landscape phages are also used as affinity reagents These landscape phages display thousands of copies of peptides in a dense, repeating pattern around the tubular capsid (Petrenko & Smith 2000) The display of these thousands of peptides gives the phage surface different characteristics The binding affinity of the landscape phages for the target is not determined

by the affinity of one of these peptides alone, but by the whole structure In cDNA phage libraries, cellular proteins are displayed on the surface of the phages These cDNA phage

libraries are frequently used for protein interaction studies (Crameri & Kodzius 2001; Pelletier & Sidhu 2001; Li & Caberoy 2010; Van Dorst et al 2010c) Furthermore, they can be used as affinity reagents in diagnostic tests to detect antibodies for cellular proteins and to diagnose autoimmune diseases