Biochip Array Technology
Katie Walker
Biochip Array Product Specialist
The Technology and Applications of Protein Biochip Array
Technology
Panels of tests, allowing for the
simultaneous multi-analyte detection of
a wide range of analytes, from a single undivided
sample
•81mm2 Solid State Device
•Assays contained in discrete reaction sites
•Single patient sample per biochip
•Multiple assays performed simultaneously
Development of Biochip Array
• 1992 – 2003• Over 185 Scientists involved• $140,000,000.00 Investment• 45 new patents• Three main stages in Biochip development
– Synthetic Chemistry– Nano Dispense– Detection technology
The Biochip• The crux of the technology is the Biochip itself
• Coated in a proprietary silanation process– Activation of the Biochip surface
• Ensures uniformity and reproducibility
– Modification of surface chemistry • Controlled anti-body binding in optimal orientation
Biochip Surface Chemistry Non -activated Surface
Activated Surface
Biochip Manufacturing
• All manufactured by Randox at our HQ in the U.K.
• 20 million chip per year capability• Fully automated clean-room facility
Nano Dispense of Antibody solution
Quality Control• Quality control is at the heart of all that Randox does.• Quality is ensured at every level of product
– Pre process• Quality components • Randox Antibodies
– In process• Manufactured to ISO 13485 • Ensures reproducibility
– Post Process• Internal QC to ensure Functionality, integrity and
stability
Quality Control
Immunoassay Formats
• Tried and trusted EIA• Sandwich immunoassay
– Cytokine and Growth Factor Array• Competitive immunoassay
– Drugs of Abuse Array• Horse Radish Peroxidase Label
Competitive Immunoassay
Y Y
SUBSTRATE
ANTIBODY
ANALYTEE
CONJUGATE
DETECTION
Signal Analysis
• Chemiluminescence• L.O.D. compared
– Fluorescence (ELISA) 2,000,000 zeptomoles– Radio Immunoassay 1,000,000 zeptomoles– Chemiluminescence 25,000 zeptomoles
» Tietz ‘Clinical Chemistry’
• Imaged with super cooled CCD camera• Quantified by custom software
Available Arrays• Drugs of Abuse Array 1• Cytokine and Growth
Factor Array 1• Free Thyroid Array• Total Thyroid Array• Fertility Array• Tumour Monitoring
Array• Tumour PSA Array• Adhesion Molecule
Array
• Cardiac Array• Growth Promoter Array• Anti-Microbial Array 1• Cardiovascular DNA
Array• Colorectal Cancer DNA
Array• Anti-Microbial Array 2*• Anti-Microbial Array 3*• SyntheticSteroidsArray*• Drugs of Abuse Array2*
Drugs of Abuse Array 1
• Amphetamine• Methamphetamine• Barbiturates• Benzodiazepine I• Benzodiazepine II• Cocaine Metabolite
• Methadone• Opiates• Phencyclidine• Cannabinoids• Creatinine (urine
application only)
Drugs of Abuse Array II
• Buprenorphine• Fentanyl• Ketamine• LSD• MDMA• Hydromorphone
• Methaqualone• Oxycodone I• Oxycodone II• Propoxyphene• Creatinine*
Drugs of Abuse Arrays
• Specific matrix optimised kits for– Urine (FDA cleared)– Urine Semi-Quant– Whole Blood Quant– Oral Fluid Quant
Amphetamine Class
50 ng/mL /50 ng/mL
25 ng/mL /50 ng/mL
1000 ng/mL /1000 ng/mL
Cut-off
d-Amphetamine / + Methamphetamine
d-Amphetamine / + Methamphetamine
d-Amphetamine / + Methamphetamine
Target
Oral FluidBloodUrine
36%0.4%MDMA
0.4%544%MDA
100%<0.2%+ Methamphetamine
1.0%100%d-Amphetamine
Methamphetamine assayAmphetamine assayCross reactivity (urine)
Barbiturates
5 ng/mL50 ng/mL200 ng/mLCut-off
PhenobarbitalPhenobarbitalPhenobarbitalTarget
Oral FluidBloodUrine
84%Amobarbital
183%Pentobarbital
172%Butalbital
40%Barbital
512%Secobarbital
100%Phenobarbital
Barbiturates assayCross reactivity (urine)
Benzodiazepines
1 ng/mL /1 ng/mL
50 ng/mL /50 ng/mL
200 ng/mL /200 ng/mL
Cut-off
Oxazepam /Lorazepam
Oxazepam /Lorazepam
Oxazepam /Lorazepam
Target
Oral FluidBloodUrine
Benzodiazepines
<0.1%512%Diazepam
1007%Lorazepam
<0.1%100%Flunitrazepam
1.2%241%Nitrazepam
2%250%Nordiazepam
<0.1%444%Temazepam
23.9%<0.1%Lorazepam glucuronide
68.7%8.4%Clonezapam
<0.1%1818%Alprazolam
2%100%Oxazepam
Lorazepam assayOxazepam assayCross reactivity (urine)
Cannabinoids
4 ng/mL10 ng/mL50 ng/mLCut-off
−9 THC11 nor −9 THC-COOH11 nor −9 THC-COOHTarget
Oral FluidBloodUrine
<5%11 hydroxy −8 THC
3%11 hydroxy −9 THC
49%11 nor −8 THC-COOH
<0.5%Cannabidiol
<0.5%Cannabinol
100%11 nor −9 THC-COOH
Cannabinoids assayCross reactivity (urine)
Methadone
5 ng/mL25 ng/mL300 ng/mLCut-off
MethadoneMethadoneMethadoneTarget
Oral FluidBloodUrine
<1.0%LAAM
<0.1%EMDP
<0.1%EDDP
100%Methadone
Methadone assayCross reactivity (urine)
Opiates
40 ng/mL25 ng/mL300 ng/mLCut-off
MorphineMorphineMorphineTarget
Oral FluidBloodUrine
17%Hydrocodone
27%Hydromorphone
13%Dihydrocodeine
67%Morphine-3-glucuronide
115%Codeine
100%Morphine
Opiates assayCross reactivity (urine)
Phencyclidine
10 ng/mL5 ng/mL25 ng/mLCut-off
PhencyclidinePhencyclidinePhencyclidineTarget
Oral FluidBloodUrine
90%TCP
100%Phencyclidine
Phencyclidine assayCross reactivity (urine)
Cocaine Metabolite
20 ng/mL50 ng/mL300 ng/mLCut-off
BenzoylecgonineBenzoylecgonineBenzoylecgonineTarget
Oral FluidBloodUrine
<0.1%Ecgonine
4.1%Cocaethylene
6.4%Cocaine
100%Benzoylecgonine
<0.1%Norcocaine
<3%Ecgonine methyl ester
Cocaine metabolite assayCross reactivity (urine)
Sensitivities and RangesAssay Type Urine Whole Blood
Sensitivity Assay Range Sensitivity Assay Range
Amphetamine 8.1ng/ml 0-2500ng/ml 0.4 ng/ml 0-125ng/ml
Methamphetamine 45.2ng/ml 0-2000ng/ml 6.25 ng/ml 0-250ng/ml
Cocaine Metabolite 2.3ng/ml 0-800ng/ml 0.01 ng/ml 0-250ng/ml
Methadone 0.6ng/ml 0-800ng/ml 0.13 ng/ml 0-125ng/ml
Cannabinoids 3.6ng/ml 0-150ng/ml 0.28 ng/ml 0-50ng/ml
Barbiturate 9.4ng/ml 0-800ng/ml 0.1 ng/ml 0-250ng/ml
Opiates 0.8ng/ml 0-3500ng/ml 0.12 ng/ml 0-125ng/ml
Benzodiazepine 1 0.6ng/ml 0-500ng/ml 0.04 ng/ml 0-250/ng/ml
Lorazepam 1.1ng/ml 0-500ng/ml 0.1 ng/ml 0-250/ngml
Phencyclidine (PCP) 1.9ng/ml 0-100ng/ml 0.16 ng/ml 0-50ng/ml
Drugs of Abuse Array IIAssay Specificity
Buprenorphine Buprenorphine
Fentanyl Norfentanyl & fentanyl
Hydromorphone Hydromorphone
Ketamine Norketamine & ketamine
LSD Nor-LSD & LSD
MDMA MDMA & MDEA
Methaqualone Free & 2’-hydroxymethaqualone, 3’-hydroxymethaqualone & 4’-hydroxymethaqualone
Oxycodone 1 Noroxycodone & oxycodone & hydrocodone
Oxycodone 2 Oxymorphone & oxycodone
Propoxyphene Norpropoxyphene & propoxyphene
Whole Blood Trials
• MEO USA trial using pre/post-mortem bloods.
• 33 samples x 10 assays = 330 tests completed.
• 1 x Investigator runs.
• Overall completion time = 1 hr 40 mins.• Includes:-
• Sample preparation (dilution ¼)• Calibrator Reconstitution• Controls
METHAMPHETAMINE AMPHETAMINE BENZODIAZEPINES
+ - + - + -EVIDENCE + 1 0 EVIDENCE + 0 1 EVIDENCE + 7 0
- 0 32 - 0 30 - 0 23
% AGREEMENT 100.0 % AGREEMENT 96.8 % AGREEMENT 100.0
METHADONE OPIATES PHENCYCLIDINE
+ - + - + -EVIDENCE + 4 0 EVIDENCE + 3 0 EVIDENCE + 1 0
- 0 29 - 0 29 - 0 32
% AGREEMENT 100.0 % AGREEMENT 100.0 % AGREEMENT 100.0
BENZOYLECGONINE OVERALL
+ - + -EVIDENCE + 10 0 EVIDENCE + 26 1
- 0 22 - 0 198
% AGREEMENT 100.0 % AGREEMENT 99.6
GC-MS
GC-MS
GC-MS
GC-MS GC-MS
GC-MS GC-MS
GC-MS
Urine Evaluation
• FDA Trials
• To assess the overall performance of drugs of abuse screening on the evidence immunoassay technology
• To compare the evidence performance to an alternate immunoassay system
• To compare the evidence performance to results obtained by GC/MS
Urine Evaluation
• More than 1300 clinical urine samples covering the entire range of possible test results for all ten drugs of abuse
• Comparison carried out by independent clinical toxicology laboratory in Texas
• 10% of samples fell within ±25% of the assay cut-off concentrations
82.1
3.2
96.8 96.7
17.93.3
0
20
40
60
80
100
evidence with CEDIA evidence with GC/MS CEDIA with GC/MS
% AGREEMENT% DISAGREEMENT
% Agreement Total number of samples Discrepant samples
evidence with CEDIA 96.8 1264 40
evidence with GC/MS 96.7 305 10
CEDIA with GC/MS 82.1 234 42
Amphetamines
Methamphetamine
82.2
3.3
96.7 97.0
17.8
3.0
0
20
40
60
80
100
evidence with CEDIA evidence with GC/MS CEDIA with GC/MS
% AGREEMENT% DISAGREEMENT
% Agreement Total number of samples Discrepant samples
evidence with CEDIA 96.7 1249 40
evidence with GC/MS 97.0 305 9
CEDIA with GC/MS 82.2 219 39
Cannabinoids
81.8
2.1
97.993.9
18.2
6.1
0
20
40
60
80
100
evidence with CEDIA evidence with GC/MS CEDIA with GC/MS
% AGREEMENT% DISAGREEMENT
% Agreement Total number of samples Discrepant samples
evidence with CEDIA 97.9 1335 28
evidence with GC/MS 93.9 231 14
CEDIA with GC/MS 81.8 231 42
Opiates
60.0
7.4
92.6 89.1
40.0
10.9
0
20
40
60
80
100
evidence with CEDIA evidence with GC/MS CEDIA with GC/MS
% AGREEMENT% DISAGREEMENT
% Agreement Total number of samples Discrepant samples
evidence with CEDIA 92.6 1334 99
evidence with GC/MS 89.1 320 35
CEDIA with GC/MS 60.0 320 128
Overall Summary
Evidence with CEDIA 96.9
Evidence with GC/MS 90.5
CEDIA with GC/MS 83.4
Total number of tests 11,750
% Agreement
User Defined Cut-off selection. Analysers and Software
Features
• Semi-automated• Based on Biochip Array Technology
– Multiple test sites per biochip• Fast Results
– 54 biochips ie. 540 Tests in 1 hour• Small sample volumes
– Urine – 3.5μl per test– Whole Blood - 6μl per test– Oral Fluid - 10μl per test
Features
• Quality of results– All Randox products are developed to ISO 13485,
diagnostic standards– Full trace-ability through manufacture– Antibody Specificity – Self sufficient antibody supply– Specific Methamphetamine and Lorazepam
assays
Features
• Cut-off selection– Cut-offs can be selected and reselected without
recalibration– Different cut-offs can be selected for the same
analyte during the same run, ie. Opiates sample A 25ng/mL, sample B 10ng/mL.
• Fully Quantitative Analysis– Identified as positive/negative – Concentration of compound also displayed
Features
• Software– Windows XP® based– Colour coding easy to follow– Multiple search formats– Easy export of results– Fully LIMS connectable– Extensive QC facilities
• Levy Jennings and Multi-point rules.
– Enhance trouble shooting
Features
• Fully Automated– Walk away system
• High throughput– 130 samples per hour x no.of tests per chip
• Chain of custody Features– Sample access doors lock once sample is loaded
• Password Protected
Features
• Retrospective Testing– Although only invoiced for tests reported, all assays
are carried out simultaneously– If certain assays are not reported they can be recalled
at a later stage.
References
Molloy RM, McConnell RI, Lamont JV, FitzGerald SP. Automation of biochip array technology for quality resultsClin Chem Lab Med; 43 (12):1303-1313
FitzGerald SP, Lamont JV, McConnell RI, Benchikh EO; Development of a high throughput automated analyser using biochip array technology; Clinical Chemistry; 51 (7) 1165-1176
www. .com