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1.1.1
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Assay File
1.1.1.1 Description---The assay file provides references to assay results including measurements, raw data files, derived data files, image files, and other file types. There can be multiple
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assay files per study.
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1.1.1. |
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2 _File Format |
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The nano-TAB assay file leverages the ISA-TAB file format, |
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which is a horizontal |
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-based spreadsheet format with column headers and row values. An example subset of this format is provided in TABLE |
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5. Please note, concepts specified within the bracket “\[ \]” are |
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variables depending on the type of assay. |
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1
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Material Sample Name
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Assay Name
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Factor Value [temperature]
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Unit
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Measurement Value [size]
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Unit
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]]></ac:plain-text-body></ac:structured-macro>
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NCL-23-F1
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Size by DLS
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25
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Celsius
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.
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nm
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NCL-23-F1
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Size by DLS
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37
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Celsius
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9.8
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nm
1.1.
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3 Nano-TAB Extensions
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---In support of nano-TAB, the
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extensions and constraints in Table 6 were applied to the ISA-TAB
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assay file and are further described in Section
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Field
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Change
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Description
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Field Status
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Measurement Value[]
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Addition
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Optional
5.6. One important extension is that nano-TAB allows the entry of summary data in the assay files through an optional field called Measurement Value, as described in TABLE 6.
1.1 Assay File
1.1.1 The assay file is a horizontally oriented spreadsheet, where the field names are organized as column headers in the first row. TABLE 23, TABLE 24, and TABLE 25, show an example of an assay file. According to the ISA-TAB specification, the assay file has different types of fields called nodes, attributes of nodes, qualifiers of nodes’ attributes, and other valid fields. The assay
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Statistic
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Addition
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The ISA-TAB format allows for the specification of units (e.g. mg/mL) but not a statistic (e.g. mean, SD, etc.).
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Optional
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Factor Value[]
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Use NPO
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The NPO should be leveraged to obtain a list of valid Factor values which are typically stressors or experimental conditions such as temperature, media solvent, time, and concentration.
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Optional
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1.1 Assay file
The Assay file is a horizontally oriented spreadsheet. The file supports the following types of information:
s the 1.1.1.1 Source or sample name;
1.1.1.2 Assay s the assay protocol reference and protocol parameter values;
1.1.1.3 Assay s the assay name, performer, and date the assay was performed;
1.1.1.4 Assay s assay factor values including units and statistics, if applicables assay ;
1.1.1.5 Assay measurement values including units and statistics, if applicable; and
1.1.1.6 Raw data files, s derived data files or image files, if applicable.
1.1.2 Specific concepts often leveraged in the Assay file are provided below.
assay file are:
1.1.2.1.1 Source Name---The unique identification name of the source referred to from within the study file. This column is used only if the source is same as the sample in an assay. Source names can be qualified only using Comment.
1.1.2.1.2 Sample Name--Sample Name - The unique identification name of the sample, which is referred to from within the study file. The sample is obtained after the application of a protocol. The sample name can be a biospecimen, nanomaterial, small molecule, or other material.
Assay Protocol Ref - The name of the protocol used to perform the experiment.
Sample names can be qualified only using Comment.
1.1.2.1.3 Image file---The name or URI of an image file generated from an assay.
1.1.2.1.4 Raw data file---The name or URI of the raw data files.
1.1.2.1.5 Derived data file---The name or URI of the file resulting from data transformation or processing.
1.1.2.2 Attributes of assay nodes---One or more attributes are used to provide more information about a assay node. The different assay node attributes are described below:
1.1.2.2.1 Material type---An attribute for the sample name (e.g., biospecimen, nanoparticle sample, small molecule etc.) if the same attribute is not given in the study file. The term can be a free-text description or taken from an ontology or a controlled vocabulary. If it is the latter, then the following qualifiers are used: term accession number and term source REF.
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*Parameter Value 1.1.2.2.2 _Characteristic\[\] _---*An Valuesattribute offor concepts_sample associatedname_ withif anthe experimentsame protocolattribute that areis not variedgiven in the experiment. Parameters concepts are variable and described in brackets. |
Performer - The full name of the individual performing the assay.
study file. |
1.1.2.2.3 Assay name---The name of the assay performed. This name is used as an identifier within the assay file.
1.1.2.3 Attributes of processing events for assay nodes--- One or more attributes are used to describe a step in the assay of a sample.
1.1.2.3.1 Protocol REF--- The name of the protocol used to perform the experiment. This name should be obtained from a value for the field study protocol name in the investigation file.
1.1.2.3.2 Performer---The name of the person who carried out the protocol.
1.1.2.3.3 Date--Date -The calendar day on which the assay was performed in the format protocol was carried out. The date format should be in YYYY-MM-DD.
Assay Name - The name of the assay performed.
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*Factor Value \[\] -* An independent variable manipulated by the experimentalist with the intention to affect biological systems (i.e. stressor). Factor concepts are variable and described in brackets. |
1.1.2.3.4 _Parameter value\[<parameter term\]_--- Value of a parameter, which is kept constant, when applying a protocol. The parameter term is written within brackets and must match the term used as value for the _study protocol parameter name_ in the investigation file. |
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*1.1.2.3.5 _Measurement Valuevalue_ \[<measurement term>\] ---* The endpoint of the assay. Measurement value concepts are variable and described in brackets. |
Unit - The standard of measurement.
Statistic - A datum that can be represented numerically.
Term Accession Number - Identification number of a term selected from an ontology or a controlled vocabulary, and entered as a value for the Unit.
Term Source Ref - Name of the ontology or controlled vocabulary from which a term is selected and entered as a value for the Unit.
This field is used to capture measurement outputs recorded in summary data. |
1.1.2.4 Qualifiers for assay nodes’ attributes---Each node attribute may be qualified using the following concepts, if applicable.
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1.1.2.4.1 _Unit_---The standard of measurement used if the values for _characteristic_\[\], _parameter value\[\]_ or _factor value\[\]_ columns are quantitative and dimensional. If the term for _unit_ is taken from an ontology/controlled vocabulary, then its _term accession number_ and the _term source REF_ should be defined in the nano-TAB investigation file. |
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1.1.2.4.2 _Term Accession Number_---Identification number of a term selected from an ontology or a controlled vocabulary, if the term is entered as a value in _source name_, _material type,_ _characteristic \[\]_, _parameter value\[\]_, _unit_ or _factor value\[\]_ columns. |
1.1.2.4.3 Term Source REF---The name which identifies the source from where a term is selected and entered in nano-TAB study files. This name should match one of the names entered in the term source name field in the nano-TAB investigation file.
1.1.2.4.4 Statistic---The type of statistical measure attributed to a numerical value (e.g., mean, standard deviation, z-average, etc.). This is a required field, if the parameter value, factor value, or measurement value is a statistical measure.
1.1.2.5 Other assay fields
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1.1.2.5.1 _Factor Value\[<factor name>\]_---The value of an independent variable manipulated by the experimentalist with the intention to affect the subject of study (i.e. stressor). Factor terms are given in brackets, and must be defined in the nano-TAB investigation file in the STUDY FACTORS section. Factor Value\[\] in assay file should reference technical variations (such as software, instrument or protocol variations). |
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1.1.2.5.2 _Comment\[\]_---Any comment that provides additional information, which is added only when no other appropriate field exists. |
1.1.3 The assay The Assay file will vary depending on the type of assay performed and protocol and technology type leveraged. Provided below in 5.6.4 and 5.6.5 are examples only of Assay assay files for some of the two common types of assays identified across nanotechnology resources.
1.1.
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4 Example Assay
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File: Size by Dynamic Light Scattering (DLS)
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Dynamic Light Scattering (DLS) ---DLS is a type of spectroscopy that utilizes uses a laser beam to irradiate a sample containing particles in suspension , resulting in light scattering. Rapid fluctuations in scattering intensity , around a mean value at a certain angle , occur because of particle diffusion and are dependent upon on particle size. The calculated correlation function yields a diffusion coefficient, for a given temperature and viscosity, which that can be used to calculate particle size. Exhibit 4.15 illustrates and example Assay file for Size by DLS.
Exhibit 4.15 Example Assay file for Size by DLS
1.1.2 Example Assay file: Zeta Potential
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TABLE 23, TABLE 24, and TABLE 25 illustrate an example assay file for size by DLS.
Exhibit 4.16 Example Assay file for Zeta Potential
1.1.3 Example Assay file: Assay file for Hemolysis
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Exhibit 4.17 Example Assay file for Hemolysis
1.1.4 Example Assay file: Assay file for Cell Viability (MTT)
1.1.5 Example Assay File: Assay File for MTT Cytotoxicity--
The MTT assay is a colorimetric assay that can assess the viability of cells by quantitation of the reduction of the yellow substrate MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a product that has a purple color. This assay can measure the cytotoxicity of a chemical or drug by determining the affect of treatment on cell viability. Exhibit 4.18 illustrates and example cell viability assay (MTT).
Exhibit 4.18 Example Assay file for Cell Viability (MTT)
1.1.5 Example Assay file: Assay file for Caspase 3 Apoptosis
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Exhibit 4.19 Example Assay file for Caspase Apoptosis
1.1.6 Example Assay file: Assay file for Pharmacokinetics
Exhibit 4.20 illustrates and example assay file for pharmacokinetics.
ExhibitTABLE 26 illustrates an example for a MTT cytoxicity assay (MTT) performed on three nanoparticle samples, where 9 dilutions of each nanoparticle sample are exposed to porcine proximal tubule cells for three different times of exposure (6h, 2h, and 48 h).
