CHAPTER 1

CHAPTER 1: INTRODUCTION
1.1: RESEARCH BACKGROUND
Marine algae are one of the members that include in the plant kingdom. It plays important roles for ecosystem of marine life. Firstly, algae provide a source of oxygen to other organisms. During the growing seasons, algae absorb and synthesize nutrients and trace metal. After that, algae are one of the photosynthetic organisms which can convert sunlight to food and energy. Then, algae as the basic aquatic food web that function as source of food and energy for other organisms in aquatic ecosystem (Marynia Kolak, 2017). Besides, certain algae also use for commercial and industrial purpose (Carla Jean McKinney, 2017). Algae are very simple chlorophyll not categories as plant that contains organisms with many cells. When algae grouped together that grow up out of control, it can cause the occurrence of a natural phenomenon which called Harmful Algae Blooms (HABs). HABs bring a harmful effect toward other organisms such as human, fish, shellfish, marine mammals and birds because it produce marine toxin under certain condition. One of the marine toxic that can cause harmful effect is Domoic Acid (DA)

Figure 1: the marine toxins
DA is one of the marine toxins produced by certain organisms. It is a marine –based neurotoxin that produced by some diatom species of the genus pseudo-nitzchia in bloom condition such as N.Pseudodelicatissma. This diatom can be found in the shellfish. According to Olga M. Pulido, domoic acid is structurally similar to the kainate acid that naturally occurs in some seaweed. This toxin was bioaccumulate through feeding on prey that feed on the algae such as shellfish and anchovies. They accumulated in those marine organisms and transfer to higher trophic levels along food chain. Domoic acid that produced by these diatom species acts as an intra and inter-species allelochemical and responsiblefor toxin algae bloom ( Emer Shannon and Nissreen Abu-Ghannam, 2016). According to emer shannon and Nissreen Abu-Ghannam, allelochemical is a process which many algae was released into the environment.
There are several that can be used to detect domoic acid in the shellfish. In this project, enzyme-linked immunosorbant assay (ELISA) method will be used as main method to detect the domoic acid that by the chromatography technique which is High performance liquid chromatography (HPLC) as instrument for validation process. ELISA is a test to determine the serum antibody concentration in a virus test. According to thermo fischer scientific, ELISA is a plate-based assay technique design to detect and quantified substance such as protein, peptides, hormone and antibodies. ELISA is useful tool since the presences of antigen, antibody concentration and amount of a substance of a solution.

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Chromatography is the process by which the components of a mixture can be separated and used for identification and qualification. One of the type of chromatography is high performance liquid chromatography (HPLC). According to Rama Rao Kadiyam, HPLC is basically a highly improved form of column liquid chromatography. This technique involves a stationary phase whether it in solid or liquid phase supported on a solid and also involves a mobile phase whether it liquid or gas.
1.2: PROBLEM STATEMENT
For many years, many people have debate about the cases of Domoic Acid Intoxication. The first case about domoic acid was discovered in the late 1980’s. According to Olga M. Pulido (2008), domoic acid was identified as the toxin responsible for an outbreak of human poisoning that occurred in Canada in 1987 following consumption of contaminated blue. Domoic acid is a chemical that is produced when the algae bloom and cause many adverse effect not only to organisms but environment too. Domoic acid poisoning bring harmful to organisms due to its characteristic structure that is composed of three carboxylic acids, a pyrrolidine ring, and (Z,E)-conjugated diene side chain. Domoic acid cause a syndrom knows as amnesic shellfish poisoning (ASP), paralytic shellfish poisoning (PSP), diarrhoeic shellfish poisoning (DSP), neurotoxic shellfish poisoning (NSP) and azaspiracid shellfish poisoning (AZP). Since then, domoic acid has been related to the several unusual mortality events involving mammals, seabirds even human also get a bad effect for those who consume shellfish because of the toxins that being produced. Symptoms of domoic acid poisoning can be seen in 30 minutes until 24 hours after eating the toxic seafood which include vomiting, abdominal cramps, headaches, disorentation, diarrhea and dizziness. (Domoic Acid and Amnesic Shellfish Poisoning Fact Sheet, 2018).
There are few factors that lead to toxin shellfish which are rising ocean temperature or global warming. According to new research by Ian Johnston, rising ocean temperature is cause to increase in shellfish in contaminated with a potentially deadly toxin. Even, the scientists have warned that if ocean temperature rises because of global warming, this could lead an increase in the number of poisonous shellfish (Ian Johnston, 2017).

1.3: OBJECTIVES
There are few objectives to achieve in this project which are:
To produce and modify the biochemical sensor using ELISA method where ninhydrin act as reagent.

To detect the Domoic acid in the shellfish using ELISA method.
To verify the result obtained from ELISA method using the chromatographic technique (HPLC-UV)
1.4: SIGNIFICANT OF STUDIES
Many people consume seafood in their daily life especially shellfish. Shellfish brings lots of benefits which is it high in protein and low in fat. After that, it also consist varies in vitamin such as vitamin A and Vitamin B and also rich in minerals including B12, Zinc, Omega-3 fatty acid and etc but after the first case happened in Canada in late 1987, domoic acid that produced by the shellfish has become major concern in society. In this project, it is very importance to detect the domoic acid in shellfish to avoid unwanted things happen to the organism especially to human. As we know, domoic acid is a poisonous toxic that can bring death to human and other organisms besides to the environment. In order to detect the analyte which is domoic acid, the indirect ELISA method will be use in this project with ninhydrin as reagent. The reasons why ELISA will be use in this project are because it suitable alternative to HPLC-UV as it offer advantages in sample turnaround and accessibility over the instrument method. Besides, it also easily gives precise and valid for DA analysis in shellfish. After that, it easily can be operate in small and regional laboratories with little investment needed and will be a practical way of identifying negative samples as part an effective Hazard Analysis Critical Control Point (HACCP) system. (Hans Kleivdal ; etc, 2007).

CHAPTER 2: LITERATURE REVIEW
2.1: DOMOIC ACID
2.1.1: DESCRIPTIVE OF DOMOIC ACID
Domoic acid with IUPAC name (2S,3S,4S)-4-(2Z,4E,6R)-6-carboxyhepta-2,4-dien-2-yl-3-(carboxymethyl)pyrrolidine-2-carboxylic acid is a toxin that produced by members of diatom genus pseudonitzchia when the algae or plankton bloom. However, this diatom also related to the amnesic shellfish poisoning (ASP). ASP is an illness that can be a life threatening syndrome that is distinguish by both gastrointestinal and neurological disorder. Domoic acid is a neurotoxin that blocking neurochemical processes, lead to short-term memory loss, brain damage and lastly its can cause death to human.
In late 1987 in Canada, 3 people was reported died after eating blue mussels from Prince Edward Island, while more than 100 persons became ill. However, the victims that still can survive and recover within 10 days after a period of confusion and nausea, but continued to show short-term memory loss (Todd, 1993),citation from Lizzy Mos, 2000. They found that the mussel was contained high level of neuroexcitatory toxin domoic acid. The source of domoic acid was detected to a bloom and also known as pseudo-Nitzschia multiseries (Bacillariophyceae) (Hasle, 1995). Since then, many cases have been described. DA actually had been discovered in the 1950s in red microalgae, Chondria armata, in Japan. In small doses, they used the domoic acid as medicine as an anthelmintic among children with no reported sign of any poisoning effect such as ASP. Anthelmintic is a group of antiparasitic drug to remove parasitic worms (helminths) internal parasit especially parasit worms from the body by stunning or killing and without bringing a harmful to the body.
Over the past 25 years, there are many cases that involve the toxication of domoic acid (Lynn M. Grattan et al., 2018). For example, measurable levels of DA have been found in the coastal waters and shellfish on the Pacific Coast of the United State and during the 2015 and 2016, the continuation of harmful algae bloom was recorded, accompanied by the longest lasting Domoic Acid levels documented in Pacific razor clams (RC, Siliqua patula), presumably triggered by warm Pacific Ocean anomalies, by McCabe, R.M.; Hickey, B.M.; Kudela, R.M.; Lefebvre, K.A.; Adams, N.G.; Bill, B.D.; Gulland, F.M.; Thomson, R.E.; Cochlan, W.P.; Trainer, V.L. An unprecedented coastwide toxic algal bloom linked to anomalous ocean conditions. Geophys. Res. Lett. 2016, 43, 10366–10376. CrossRef PubMed 15. McKibben, S.M.; Peterson, W.; Wood, M.; Trainer, V.L.; Hunter, M.; White, A.E. Climatic regulation of the neurotoxin domoic acid. Proc. Natl. Acad. Sci. USA 2017, 114, 239–244. CrossRef PubMed

Figure 2.1: the chemical structure of domoic acid
2.1.2: PROPERTIES OF DOMOIC ACID
Domoic acid is present in white solid with colourless crystal needle. It has melting point around 213ºC – 217ºC that soluble in water. After that, it also can be dilute in mineral acids and alkali hydroxide solution. It is slightly soluble in methanol and ethanol but insoluble in petroleum ether and benzene. Domoic acid is a quite small molecule with a molecular weight of 311.33 g/mol. According to Sigma Adrich, Domoic Acid is stable under certain recommended storage conditions. It emits toxic vapors of nitrogen oxides when heated to decomposition.
Properties Chemical formula C15H21NO6
Molar mass 311.334g/mol
density 1.27 g/cm3
Vapour pressure 1.5 × 10-11 Hg (25ºC)
Solubility (water) Soluble in water (8mg/ml)
log Kow -0.89
2.1.3: FACTORS OF DOMOIC ACID
There are several factors that lead to increasing intoxication domoic acid which are global climate change, eutrophication that came from uses of fertilizers. Then, agriculture and urban runoff also main contributor to domoic acid. After that, aquacultures activities and coastal development also main factors of domoic acid.2.1.4: SIDE EFFECTS OF DOMOIC ACID TOWARDS HUMAN
Domoic acid can brings lots of effect toward humans. It can affect the human’s brain and cause fatigue, hallucinations, abnormalities, seizures and may result in death. The toxins can attack a part of the brain called hippocampus. Hippocampus is a small organ located within the brain’s medial temporal lobe and part of the limbic system that responsible for emotions, associated mainly with memory and long term memory. (Ananya Mandal, 2014).Hippocampus works to unite and organize new memory, connects emotions and sense to memories and also helps in navigation and orientation. After that, it also causes nausea and vomiting due to ASP syndrome and even
2.2: DESCREPTIVE OF MARINE TOXIN
Marine toxin is produced by various species of algae under certain condition. Marine toxin especially shellfish toxins (phytotoxins) are the most dangerous marine biotoxins. Shellfish that contaminated by marine biotoxins usually looking normal and good from the outside even their smell and taste are nice but once they consume it, they possibly suffer from several of gastrointestinal and neurological illness. The domoic acid poisoning first recorded as amnesic shellfish poisoning (ASP).

2.2.1: Amnesic shellfish poisoning (ASP)
In late 1987 in Canada, Amnesic shellfish poisoning (ASP) cause by domoic acid was discover for first time that involve shellfish reported in density of the pennate diatom pseudo-nitzschia multiseries near up to 15×1016cells/L. Over 200 cases of mussel related illness which involves 107 people in total. 47 of them were men while the rest were women. In addition, 49 were between 40 and 59 years old and 38 patient were 60 years or older. (Olga M. Pulido, 2008). The symptoms of ASP can appear in 24 hours after someone consume the affected shellfish. They may involve diarrhea, abdominal cramps, vomiting, nausea, haemorrhagic gastrists, and memory loss. The memory loss symptoms that intoxicated with ASP is kind a similar with Alzheimer’s disease.
According to Hong-Chang Lim et al, 2010. In the same years, 12 cases of human health were reported along the Pacific coast of Washington and Oregon where shellfish such as mussels and oyster were found to contain up to 154?g/g of domoic acid.
2.2.2: paralytic shellfish poisoning (PSP)
PSP is one of four syndromes that caused by shellfish poisoning. PSP’s syndrome usually begin minutes or hours after consume contaminated shellfish by ingestion. There few symptoms that same with the ASP’s symptoms include vomiting and adding with new symptoms which are shortness of breath, choking feeling, confused and even loss of coordination. There is no anti-toxin for shellfish poisoning. According to Washington State department of health, the sources of PSP are bivalve mollusks such as clams, oyster and mussels concentrated PSP toxin and DSP toxin. There is no person-to-person spread.
2.3: ENZYME-LINKED IMMUNOSORBANT ASSAY (ELISA)
2.3.1: Descriptive of ELISA
ELISA is an alternative method to the current liquid chromatography for Domoic Acid determination. As it was mentioned above, according to Thermo Fischer Scientific, ELISA is a plate-based assay technique design to detect and quantified substance such as protein, peptides, hormone and antibodies. It is a biochemistry assay that uses a solid phase enzyme immunoassay (EIA) as it employs an enzyme linked antigen or antibody as a marker to detect a substance which is an antigen in a sample. According to Biobest website, antibody is made to give the feedback to infection so antibody ELISA can show whether the sample was contaminated with certain virus or not. Elisa is used as a diagnostic tool in medicine, plant pathology and as quality-control checker in a various industry. ELISA is faster, highly sensitive, highly throughput, reproducible, and flexible with the ability to analyze a variety of different sample types of diverse origins. It is a test that uses antibodies and color change to identify a substance. ELISA is divided to four types which are direct ELISA, indirect ELISA, sandwich ELISA and Competitive ELISA. In this project, Indirect ELISA will be use as an alternative method to detect the Domoic Acid.

Figure 2.3.1: the microtiter plates of ELISA
2.3.2: Application of indirect ELISA
Indirect ELISA is a two step ELISA that involved two steps binding process which use primary antibody and secondary labeled antibody. In this technique, the primary antibody is incubated with the antigen-coated wells. Then, the labeled secondary antibody is added. This secondary antibody is a polyclonal anti-species antibody. After that, the substrate is added to the well to produce signal amplification. Indirect ELISA detection is versatile as different visualization markers can be used with the same primary antibody.

Figure 2.3.2: the procedure of indirect ELISA
2.3.3: Advantages of indirect ELISA
There are many advantages of using Indirect ELISA as main method to detect domoic acid in shellfish such as it has high sensitivity compare to other method because labeled secondary antibody can bind to the each primary antibody that contain several epitopes, allowing for signal amplification. After that, it has great flexibility because different primary detection antibodies can be used with one labeled secondary antibodies. Then, it cost saving because fewer labeled antibodies are require. Besides, maximum immunoreactivity of the primary antibodies is maintained because it is not labeled.
2.3.4: Disadvantages of Indirect ELISA
There are disadvantages of indirect ELISA such as in this procedure, an extra incubation is needed for secondary antibody and cross-reactivity might occur with the secondary antibodies, resulting in nonspecific signal or possibility to produce a background noise.

2.4: REAGENT: ninhydrine
2.4.1: descriptive of ninhydrinNinhydrin reagent with IUPAC name (2,2-Dihydroxy-1H-indene-1,3(2H)-dione) was discover by Siegfried Rumemann, the German-english chemist in 1910 that observed the reaction between ninhydrin with amino acids. Ninhydrin is a stable hydrated product of indane-1,2,3,-trione which used in chemical assay for peptide bonds such as in protein determination. It used to detect ammonia or primary and secondary amines. When ninhydrin react with primary or secondary amines, it produces a blue or purple reaction product (diketonehydrindyliene-diketonehydrinediamine). According to Li Liang, It can be categories as the most important and widely used reagent to detect and analyze amino acids because amino acids link together via amide bond with carboxylic acids amine group forma chains. Ninhydrin as a peripheral membrane protein, since the protein sequence are highly hydrophilic and contain many charged amino acid.

Figure 2.4.1: the chemical structure of ninhydrin2.4.2: properties of ninhydrinNinhydrin is a chemical that present in a white to light yellow crystal or powder form and it becomes anhydrous with reddening at 257-266ºF. Ninhydrin is odorless colourless to cream coloured crystalline solid. It’s soluble in ethanol and acetone at room temperature and also in water. The melting point of ninhydrin is slightly high which is 466-469ºF.properties Chemical formula C9H6O4
molecular weight 178.14
melting point 466-469ºF
solubility 1-5mg/mL at 68ºF
decomposition 241ºC
Table 2.4.2: the properties of ninhydrin
Figure 2.4.2: the ninhydrin in solid phase
2.5: CHROMATOGRAPHY TECHNIQUE
Chromatography is a technique in which the components of a mixture are separated based on differences in the rates at which they are carried through a fixed or stationary phase by a gaseous or liquid mobile phase. It is a widely used method for the separation, identification, and determination of the chemical components in complex mixtures. The stationary phase is actually phase that is fixed in place either in a column or on a planar surface, while the mobile phase is a phase that moves over or through the stationary phase carrying with it the analyte mixture. The mobile phase may present in solid, liquid or gas form.
2.5.1: High-Performance Liquid Chromatograpy (HPLC)
HPLC has become an indispensable analytical tool that most versatile and widely used type of elution chromatography. This technique is used to separate and determined the species in variety of organic, inorganic and biological materials. The mobile phase is a liquid solvent containing the sample as a mixture of solutes. Liquid chromatography can be performed in 3 primary approaches. Sample must be dissolve into a liquid that is then transported by the solvent onto or into the chromatographic device.
The components that consist in the HPLC for validation process to detect the domoic acid in shellfish are solvent, pump, single injector, columns, detectors and data collection devices. In solvent reservoir involves a stationary phase and a mobile phase whether in the gas or solid form. The mobile phase flows from solvent reservoir to HPLC pump. A pump takes the mobile phase and forces it through the system’s column and detector in liquid phase. Sample injector provides injection of the liquid sample within the range of 0.1 to 100 ml of volume with high reproducibility and under high pressure. After that, the sample move from injector into the HPLC column that filed with stationary phase with a particle size of 3-10?m. After that, the detector will detect the analyte as they elute from chromatography column. The detector will produce the signal on chart records then store then reprocess the chromatography data. Lastly, the computer will integrate the respons of the detector to each components and place in the chromatograph.

Figure 2.5.1: the schematic diagram of HPLC
2.5.2: UV-Vis spectrophotometryThe construction of a traditional UV-VIS spectrometer is similar to an IR, and has similar functions which are sample handling, irradiation, detection and output are required. The diagram below shows the simple schematic that covers most modern UV spectrometers:

Figure 2.5.2: the schematic diagram of UV-Vis spectrophotometryThere are 5 main components that consist in the UV-Vis for measuring the absorption of UV-VIS which is a source consists of UV and visible, wavelength selector (monochromator), sample container, detector and signal processor and a readout device. The sources of UV radiation are very important to make sure the power of the radiation source does not change abruptly over are its wavelength range. Then, the mechanisms for electrical excitation of hydrogen at lower pressure produce the continuous UV spectrum. Two atomic species and ultraviolet photon brake up due to a mechanism for electrical excitation.

Then, for the source of visible radiation, which is a source of visible light is consists of tungsten filament lamp. The lamp is usually used in the wavelength range between 350 nm until 2500 nm. The energy output stable when energy emitted by a tungsten filament lamp is proportional to the fourth power of the operating system and the voltage of the lamp must be very stable indeed. Tungsten or halogen lamp consists of small amount of iodine in a quartz envelope which also contains the tungsten filament. Undergo sublimation process, the iodine reacts with gaseous tungsten produce the volatile compound. After that, for wavelength selector ( monochromator), it consists of several components which are an entrance slit, collimating lens, dispersing device, focusing lens an exits slits.

Virtually all UV spectra are recorded solution-phase. Cells can be made from plastic, glass or quartz. Only quartz is transparent in the full 200-700 nm range while plastic and glass are only suitable for visible spectra. Concentration is empirically determined.
CHAPTER 3: METHODOLOGY
3.1: chemicals and reagents
Domoic acid will purchase from Sigma Aldrich, St. Louis, MO, USA. …….. Polystyrene microtiter plates, MaxisorpTM will purchase from NUNCTM (Roskilde, Denmark)….. Washing solution which is Phosphate Buffer Saline (PBS), coating buffer which are Phosphate, Buffer Saline (PBS), Bicarbonate or Carbonate and Blocking solution which are 5% non fat dry milk or PBS will purchase from……. All the reagents and chemicals will purchase from…ninhydrin reagent will be purchased from sigma aldrich, US.

3.2: Preparation of ninhydrin reagent
Weight 8g of ninhydrin and dissolve in 100ml of acetone or distilled water or coating buffer which is PBS.
3.3:

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