ANALYTAL CHEMISTRY IN POINT-OF-CARE (POC) DIAGNOSTICS Doc. RNDr. Petr Kubáň, Ph.D. CEITEC Masaryk University Bioanalytical Instrumentace petr.kuban@ceitec.muni.cz Point-of-care (POC) diagnostics Diagnostic technologies that allow analysis of patients samples outside the clinical laboratory (doctors office, at bedside, in ambulance vehicles etc.) Usually small, simple instruments/devices that are able to analyse important biomarkers in clinical samples (blood, saliva, urine) The time of analysis is decreased, no need to transport the samples. POC diagnostika - terminologie analýzy na místě (point-of-care testing) analýzy mimo laboratoř (off-site testing) analýzy u pacienta ( near patient testing) BREATH ANALYSIS Hippokrates (460 BC – c. 370 BC) fetor oris and fetor hepaticus in his treatise on breath aroma and disease fetor oris = halitosis (anaerobic bacteria in the mouth) fetor hepaticus = breath of the dead (liver failure) Hippocrates, The Corpus: The Hippocratic Writings, Kaplan Publishing, New York, 2008. SOME HISTORY URINE WHEEL – POC diagnostics in 15th century 1506 by Ulrich Pinder Epiphanie Medicorum Urine testing - uroscopy sweet - diabetes brownish - jaundice/hepatitis Red, foam - kidneys blood - urinary tract cancer SOME HISTORY http://health.ucsd.edu/news/features/Pages/2014-04-21- colors-that-suggest-urine-trouble.aspx Amber or honey. Possibly dehydrated. Light orange. Possibly dehydrated, but may also be caused by liver or bile duct problems Dark orange or brown. A possible symptom of jaundice, rhabdomyolysis or Gilbert’s syndrome Red.Blood in the urine, called hematuria, possible lead or mercury poisoning Blue. metabolic disorder known as familial hypercalcemia or “blue diaper syndrome” Dark brown or black. White or milky. No color. Possibly long term kidney disease or uncoltrolled diabetes. 1897 Nebelthau - those with diabetes have acetone in their breath 1874 Anstie – there is alcohol even in breath 1927 Bogen first Alco Tester McNalley Breathalyzer 1936 Patent Harger Dräger EXAMPLES OF POC DEVICES Single use/disposable Manual Portable Laboratory (portable) TYPES OF POC DEVICES Indicator papers (analytes in urine, pH, etc.) Paper microfluidics Lateral flow assays Microfluidic analyzers Smartphone based devices http://www.nanowerk.com/spotlight/spotid=21383.php IDEAL POC DEVICE INDICATOR PAPERS Suitable range indicator papers (pH 5.0 – 9.0) are used for approximate pH measurements, for instance to detect the acidification of an organism Abnormal: Some foods (such as citrus fruit and dairy products) and medicines (such as antacids) can affect urine pH. A high (alkaline) pH can be caused by severe vomiting, a kidney disease, some urinary tract infections, and asthma A low (acidic) pH may be caused by severe lung disease (emphysema), uncontrolled diabetes, aspirin overdose, severe diarrhea, dehydration, starvation, drinking too much alcohol, or drinking antifreeze (ethylene glycol). Normal: 4.6-8.01 Analytes: WBC nitrite proteins pH blood ketones urobilinogen bilirubin glucose liver diseases diabetes Urinary tract infection ketonuria hematurai Diabetes monitoring PREGNANCY TEST hCG – human choriogonadotropine – hormone hCG was discovered in the urine of pregnant women in 1927 and today’s detection in majority of the pregnancy tests relies on detection of this compound hCG glykoproteine hormone hCG is formed from 2 subunits, α a β α subunit of hCG = non-specific β subunit of hCG = specific hCG can be determined in urine hCG levels double about every two days in a pregnant woman, so the test is much more reliable two weeks after conception than one week later. The tests work by binding the hCG hormone, from urine, to an antibody and an indicator. The antibody will only bind to hCG; other hormones will not give a positive test result. The usual indicator is a pigment molecule, present in a line across a home pregnancy urine test. The most sensitive tests are able to detect 10 mIU/ml, usually though 20mIU/ml. Best if performned in the third week of suspected pregnancy. The blood test can detect already 2 mIU/ml. LATERAL FLOW ASSAYS (LFA) Series of membranes, overlapping Material: cellulose, nitrocellulose, cellulose acetate, glass fibres, etc. LATERAL FLOW ASSAYS (LFA) SAMPLE PAD – sample introduction (pipette) – alternative blood plasma separation (to remove the red blood cells) – uniform flow of the sample CONJUGATE PAD – contains antibodies specific for the analyte – antibodies are bound to particles that can be detected – blue color – latex particles (nm size)modrá barva – latexové částice (velikost n – red color - Au particles MEMBRANE – different material, usually hydrophobic nitrocellulose – two zones on the membrane : control and test – test zone contains immobilized antigens that can capture the analyte with the bound color particle ABSORBENT PAD – control zone – to test the functionality of the LFA – usually cellulose – keeps constant flow of reagents and sample – collection of waste PREPARATION OF LFA STANDARD LFA Protilátka Stanovovaná látka Au nanočástice COMPETITIVE LFA AIDS TEST (2012) VISITECT® HIV 1/2 is a qualitative test for the detection of antibodies to HIV 1/2 virus in human serum, plasma and whole blood specimens. The test is easy-to-use, offers high sensitivity and specificity and produces accurate results within 15 minutes. VISITECT HIV 1/2 http://www.omegadiagnostics.com/products/by-area/hiv/ AIDS TEST (2012) Omega Diagnostics Group has announced the launch of a ground-breaking new point-of-care disposable test for the detection of HIV. VISITECT CD4 detects CD4 T-cell levels and was developed by the Burnet Institute in Melbourne, Australia. An easy-to-use semi-quantitative lateral flow test, it uses a finger-prick blood sample to produce a visual result in 40 minutes, enabling patients to receive life-saving antiretroviral treatment before leaving the clinic. Yen et al. Lab Chip, 2015, 15, 1638 EBOLA, DENGUE FEVER, YELLOW FEVER TESTS C-reactive protein (CRP) is a protein that increases in the blood with inflammation and infection as well as following a heart atack, surgery, or trauma. Normal concentration in healthy human serum is between 5 and 10 mg/L, increasing with aging. Higher levels are found in late pregnant women, mild inflammation and viral infections (10–40 mg/L), active inflammation, bacterial infection (40– 200 mg/L), severe bacterial infections and burns (>200 mg/L). The CRP level in blood increases with bacterial infections already after 6-12 hours from infection. CRP test CRP pentamer v krevní plasmě GLUCOMETERS Device for measurement of glycemia at home (self monitoring) Depending on the level of glycemia and previous experience one can estimate the amount of insulin that is necessary The goal is to achieve the glycemic curve of a non-diabetic person Glucometers can often calculate weekly and monthly averages from the measured values. Typically electrochemical methods The test strip contains narrow capillary that sucks the blood inside. The glucose is oxidized and H2O2 is released. The more glucose the more H2O2 H2O2 is electrolytically decomposed, electrons are measured as electric current and recalculated to the glucose content. https://www.accu-chek.cz/ ACCU CHECK (Roche) EXAMPLE – USE OF GLUCOMETER FOR OTHER PURPOSE Xiang, Lu, Nature chemistry, 3,2011, 697-703 Analysis of kokainu, interferon gamma, ions The method is based on the targetinduced release of invertase from a functional-DNA–invertase conjugate. The released invertase converts sucrose into glucose, which is detectable using the meter. PAPER MICROFLUIDICS http://gmwgroup.harvard.edu/ A paper-based electrochemical sensor that can be read with a commercial glucose meter. (A) Wax printing allows the high-throughput production of devices with hydrophobic regions and screen printing is used to add electrodes on top of the printed devices. (B) Comparison of a commercial test strip for glucose with a paperbased test strip. (C) The paper-based test strip fits into a commercial glucose meter. GLUCOMETER WITH PAPER SENSOR A low-cost, rapid test for sickle cell disease. After centrifugation over an aqueous multiphase system, blood cells from a donor with sickle cell disease separate by density. Dense cells characteristic of sickle cell disease form a red layer at the bottom of the test. Sickle cell anemia The average life span of people with sickle cell anemia is 42 for men and 48 for women The change of shapes of RBC A universal mobile electrochemical detector (uMED). The uMED device (left) can perform a variety of electrochemical analyses on commercial and custom electrodes. Results can be transferred through the audio jack of any phone to enable connectivity (right). A peak flow meter is commonly used by a person with asthma to measure the amount of air that can be expelled from the lungs. If the airways become narrow or blocked due to asthma, peak flow values will drop because the person cannot blow air out of the lungs as well. A peak flow meter can be a useful aid in monitoring a person's asthma over time and can also be used to help determine how well a patient's medications are working. BREATH MONITORING NO MONITORING www.nioxmino.com, www.aerocrine.com ASTHMA http://www.ncbi.nlm.nih.gov/pubmedhealth/aboutnlm/ Astma – a chronic disease that affect the lungs and the airways by narrowing the bronchioles. It is characterized by variable and recurring symptoms, reversible airflow obstruction and bronchospasms. There is no cure for asthma. Symptoms can be prevented by avoiding triggers, such as allergens and irritants, and by the use of inhaled corticosteroids. In 2015, 358 million people globally had asthma, up from 183 million in 1990. LUNG CANCER Non-small-cell lung carcinoma (NSCLC) and Small-cell lung carcinoma (SCLC) NSCLC typically surgery SCLC chemotherapy The most common cancer in the world 38 BREATH ANALYSIS Some analytes can be analyzed by CE-C4D, some by CE-LIF Average person exhales between 10-90.000 L of air per day 39Breath Research Institute of the Austrian Academy of Sciences Antoine Lavoisiser 1784 BREATH ANALYSIS 41 EXHALED BREATH CONDENSATE(EBC)– analysis of single exhalation Price: 0,05 EUR Volume of EBC/1 breath: 20 µL Price: 20 EUR Volume of EBC/1 breath : 25 µL 42 EXHALED BREATH CONDENSATE (EBC) 43 1.5 2 2.5 3 3.5 4 Time (min) C4Dsignal(mV) 5 mV A B C D E F ClNO2- NO3SO42- acetate propionate butyrate 5 consecutive exhalations, analysis by CE 1.5 2 2.5 3 3.5 4 Time (min) C4Dsignal(mV) 5 mV A B C D E F NH4 + K+ Ca2+ Na+ Mg2+ 44 PORTABLE CE INSTRUMENT B HV C4D T CP DAS INJ + G EBC ANALYSIS STD CF H Gregus et al, Journal of Breath research, 2015. 46 Glutathione (GSH) in EBC 1000x lower LOD than C4D !! 47 Glutathione (GSH) in saliva CF – cystic fibrosis Cystic fibrosis (CF) is a genetic disorder that affects mostly the lungs, but also the pancreas, liver, kidneys and intestine There is no known cure for cystic fibrosis. Lung infections are treated with antibiotics, which may be given intravenously, inhaled, or by mouth. Sometimes, the antibiotic azithromycin is used long term. Inhaled hypertonic saline and salbutamol may also be useful Half of the patients in the Czech Republic has a life span of 32 let. It is estimated that every 25th person is a carrier. It is caused by the presence of mutations in both copies of the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein 49 CF DIAGNOSTICS DIAGNOSIS: genetic disorder impaired ion transport breathing problems, lung infections, GI problems ELEVATED CHLORIDE IN SWEAT 50 SWEAT TEST ALTERNATIVE SKIN WIPE PAINFULL USE OF CHEMICALS ON THE SKIN (pilocarpine) TEST DURATION ABOUT 30 MINUTES PAINLESS NO CHEMICALS (DI WATER) TEST DURATION ABOUT 10 SECONDS 51 CE-C4D SIMULTANEOUS SEPARATION OFANIONS AND CATIONS CF 1 CF 2 H 52 USE OF ION RATIOS vs. CHLORIDE CONCETRATION PCA IN CF DIGNOSTICS -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 H1 H2 H3 H4 H5H6 H7H8 H9 H10 CF1 CF2 CF3 CF4 CF5 CF6 CF7 CF8 CF9 CF10 CF11 CF12 CF13 CF14 CF15 CF16 healthy individuals pediatric CF patients adult CF patients Principalcomponent2(33.1%) Principal component 1 (65.9 %) 54 CAPILLARY ELECTROPHORESIS (CE) IN DIAGNOSIS OF INTOXICATIONS FAST SMAL SAMPLE VOLUMES HIGH RESOLUTION HV POTENTIAL + - METABOLISM OF METHANOL CE 56 POSSIBLE TO DETECT METHANOL WITH ALCOTESTER? NEED FOR INVASIVE SAMPLING – BLOOD/SERUM PATIENT INTOXICATED WITH METHANOL UNDER VENTILATION NO FORMATE IN BREATH CONDENSATE 58 CE SEPARATION OF BLOOD PLASMA OF METHANOL INTOXICATED PATIENT MONITORING OF HAEMOLYTIC TREATMENTstandard patient haemolytic treatment PORTABLE METHANOL ANALYZER Able to determine the concentration of methanol and HCOOH in 5- 10 min Analysis from one drop of blood serum(1-10 uL) Simple use, for instance in the emergency vehicles, at doctors office. ETHYLENGLYCOL INTOXICATIONS occurs usually as unitentional ingestion of antifreeze (small children, pets) Symptoms not easy to distinguish from other intoxications ETHYLENE GLYCOL INTOXICATION 62 CE ANALYSIS OF BIOLOGICAL SAMPLES CONCLUSIONS ANALYTICAL CHEMSITRY FINDS WIDE APPLICATION IN POC DIAGNOSTICS SIMPLICITY, SPEED USE NEAR PATIENT, PORTABLE DEVICES JE STÁLE CO OBJEVOVAT 