abstract

• paper test card engineered to perform an iodometric titration
  • iodometric titration- type of redox rxn with iodine where appearance of iodine indicates end point
• card requires storage + mixing on demand of diff incompatible reagents
• titration activated when applied solution to card (dried reagents reconstituted + combined through surface-tension-enabled mixing (STEM)
• iodine atoms quantified from iodate in aq solns (0.8-15 ppm)
  • useful to quantify iodine in salts (example)
• accuracy = 1.4 ppm
• precision = 0.9 ppm
• detect B-lactam antibiotics when using iodometric back titration
• meets ASSURED criteria - to use in low resource settings where lab based analytical procedures are not very available

intro

• paper analytical devices (PADs) - good tools to solve chem problems in low resource settings
  • enable chem to be performed in locations that lack reliable access to lab reagents, glassware, instrumentation, etc.
  • they also store reagents + separate fluids /things + can act as timers
  • can be used to create/perform complex analytical tasks (like PH titrations)
  • used for quantification encournters for many technical challenges to overcome in order to have reliable measurements
    • reagents stored on paper substrate must be compatible + stable for long time
  • interest - to use paper test cards (the PADs) to address analytical need of salt producers in the developing world. salt for consumption is usually fortified w/ potassium iodate (from 15-50 ppm iodine) many of the salt producers do not have access to a titration lab to perform the iodometric titration used to assay the iodate -> the test card is designed to carry out the entire analytical task
  • used salt w/ iodate in it as a model system to establish the analytical metrics of the redox titration
  • analysis w/ iodometric titration -> xs iodide react w/ iodate in presence of acid = form triiodide
  • then triiodide titrated w/ thiosulfate using starch solution (THE INDICATOR)
  • the endpoint is reached when the color turns clear
  • show versatility of this titration device by also quantifying beta-lactam antibiotics via iodometric back titration

results + discussion

REAGENT STORAGE AND SURFACE TENSION ENABLED MIXING (STEM)

• reagents for the iodometric titration of iodate cannot be stored together on paper substrate
  • at acidic pH, iodide oxidized by air and the starch indicator is degraded by both acid and iodine (this happens both in solution AND in DRIED reagents on paper) - conclusion: strict isolation of stored reagents is necessary
• wax patterning - via rapid printing methods (whiteside) = make isolated zones on paper to separate the reagents + recombine during titration
  • loading zones made where the reagents are put and dried for storage
• after test soln added to rxn area -> card shaken
• wax barriers + solution meniscus confined the sample and reagents
• this entire process = surface tension enabled mixing (STEM)

part per million on paper

• regular iodometric titration - aloquots thiosulfate titrated onto sample until eq. point
• but on paper test card: zones in each rxn are loaded w/ diff amounts of sodium thiosulfate along w/ excess potassium iodide, tosic acid, and starch indicator in central indicator area
• iodine also stabilized more for storage as a Cd(II) salt
• aliquots applied to each rxn area to activate the titration
• paper test card not like traditional titration(which usually perform best w sharp end point indicator) - instead, each rxn area gives no color unless amount of iodate in soln overwhelms the amount of thiosulfate on rxn area
• = papercard combines features of limit tests and colorimetric quant. assay
• negative control - contains only acid + starch (should have no response when test soln applied
• iodine is detected with starch indicator
• the limit test detects iodide in concentrations greater than 20 ppm (diff squares detect diff limits of iodide)
• square 12 - positive control w/ potassium iodate, potassium iodide, tosic acid, starch to produce blue response no matter what test soln is applied

CALIBRATION OF DEVICE

• quantification by color measurement is challenging if variations captured in the sun, shade, or various lightings -> so images captured in a light box
• color measured by computer image analysis
• SD of blank samples, LOD determined to be 0.8 ppm and lower LOQ was estimated to be 2.4 ppm

INTERNAL VALIDATION

• experts analyzed solns blindly, and read w accuracy of 0.5 ppm and precision of 0.5 ppm
• range 0-75 ppm
• 2 newly trained users analyzed same 110 images, achieved accuracy of 1.4 ppm + avg precision of 0.9 ppm
• computerized image analysis -> intensities of colors measured + conc measured with calibration curves
  • 4 of the salt pads gave discrepant results + could not be quantified
• avg accuracy for image analysis by computer = 0.9 ppm
• main source of inaccuracy is systematic underestimation of the iodine concentration at high iodine concentrations
• camera software - source of error
• avg precision for range 0-15 ppm was 0.9 ppm
• avg precision for 0-7 ppm was 0.3 ppm
• = these measurements show that computer image analysis is more accurate and precise than visual estimates of concentrations by newly trained users but also that expert readers can surpass the accuracy and precision of the computer image analysis program
• visual interpretation:
  
  • advantages - increases usability in low resource settings = capture data w/ cell phone = interpret electronically = preserve it empower monitoring agencies to track quality of salt in diff geographical areas over time
  • disadvantages - human error in reading / recording results

cont.

robustness

• tested to see how storage + diff water sources affected response
• 8 ppm I standard using tap water w/ high mineral content
• hard water standards had 8% error
• lake water standards had 17% error

cont

• utility of test card for other analyses: iodometric back-titration of amoxicillin
• iodometric titration
• saw procedure for quantification of beta-lactam antibiotics (ensure quality of pharmaceutical product)
• studied if paper test card analysis could be useful for quality testing in low resource settings where analytical chem labs are rare
• tested amoxicillin conc. 0-0.9 mg/ml
• test card response showed good differentiation
• shows that cards could be used to detect antibiotics that contain 83% labeled API

cont.

suitability for use in low-resource settings

• ASSURED (affordable, sensitive, specific, user friendly, rapid, robust) criteria to assess devices meant for use in low resource settings
• considered in design
• reasons how/why:
  • complete process = 0.09$ + $.5 labor + could be decreased
  • paper test card performs iodine analysis w/ accuracy + precision that rivals other technology used in salt fortification plants in the developing world + can perform iodometric back titration w/ to detect substandard antibiotics sufficient accuracy
  • takes 5 mins
  • test cards can withstand a lot of conditions
  • test cards can be analyzed using computer image processing of mobile phone photographs -> mobile phone network for data collection + archiving

experimental section

materials

• 2% starch indicator
• p-toluenesulfonic acid
• potassium iodide
• cadmium chloride
• sodium nitrite
• anhydrous sodium thiosulfate
• secondary standard of potassium iodate
• NaCl
• amoxicillin
• iodine soln
• etc.

fabrication of saltPAD

Running + analyzing test cards

• create test soln, dilute 3.25 g salt into 15 g water (1:5) dilution
• 125 microL soln per rxn zone on the test cards + shake

SaltPAD calibration curves

• iodate standards from 0-15 ppm I in 3.7 M NaCl created by dilution from stock potassium iodate soln
• standards pipetted on saltPADs and blue color PAD respondse measured w/ imageJ

internal lab validation

• 3.7 M NaCl brine spiked with 11 levels of iodate + coded samples analyzed bind by 2 operators - each apply test samples

stability testing

- accelerated aging of test cards stimulated by storing at 40 deg celcius

back titration to quantify amoxicillin

• amoxicillin conc. from 0-1.0 mg/mL to 2.0 mL of each soln, 1.0 M NaOH
• with 2.0 mL HCl
• add 10 ml of 0.0050 M
• triiodide pipetted into soln
• diluted soln 2:25 w/ water
• new calibration curve for iodine response created bc of matrix change from salt soln to water soln

conclusions

• paper card that stoers multiple reagents + recombines them thru surface tension enabled mixing
• paper analytical device performs accurate + precise measurments of iodate in iodized salts
• cards can be used for other assays of redox active analytes
• when did for beta-lactam - gave good differentiation among amoxicilllin

Advantages to using PADs / the paper test card engineered in the experiment

enable chemistry to be performed in locations that lack reliable access to lab reagents, glassware, and instrumentation

they are also cheap to use/produce and can be done in about 5 minutes. (time/money)

can perform complex analytical tasks (like titrations)

what is the issue / topic being addressed in the paper?

using the paper test cards to address an analytical need of salt producers in the developing world

salt for consumption is usually fortified w/ potassium iodate (from 15-50 ppm iodine)

many of the salt producers do not have access to a titration lab to perform the iodometric titration used to assay the iodate -> the test card is designed to carry out the entire analytical task

CONCLUSION: TOPICS THAT COULD DEFINITELY BE ASKED BASED ON THE PAPER

• titrations
• ppm
• precision, accuracy
• dilution question
• back titration w/ iodine
• external standard
• slope and sensitivity
• LOD
• LOQ/LLOQ
• SD (std deviation)
• grubbs test

what is the indicator used

• then triiodide titrated w/ thiosulfate using starch solution (THE INDICATOR)
• in analysis:
  • excess iodide react w/ iodate in presence of acid = form triiodide
  • triiodide titrated w/ thiosulfate using starch soln as an indicator
  • if triiodide exceed reducting capacity of thiosulfate, then the indicator is BLUE
  • if its smaller than the capacity, then indicator is UNCOLORED

what is the negative control

• negative control - contains only acid + starch (should have no response when test soln applied

what is the positive control

square 12 - positive control w/ potassium iodate, potassium iodide, tosic acid, starch to produce blue response no matter what test soln is applied

why is quantification by color challenging? what can be done to overcome this challenge?

• quantification by color measurement is challenging if variations captured in the sun, shade, or various lightings -> so images captured in a light box

question abt SD, LOQ, LOD

SD of blank samples, LOD determined to be 0.8 ppm and lower LOQ was estimated to be 2.4 ppm

advantages and disadvantages to visual interpretation

• = makes it accesible
• visual interpretation:
  
  • advantages - increases usability in low resource settings = capture data w/ cell phone = interpret electronically = preserve it empower monitoring agencies to track quality of salt in diff geographical areas over time
  • disadvantages - human error in reading / recording results

diff matrixes used?

using diff water sources to test robustness of PAD

what was back titrated in the paper? What was the purpose?

iodate

• fortified salt w/ iodate in model system to carry out the analytical task of measuring iodine in salt
• redox titration
• excess iodide + iodate + acid = triiodide
• then titratef w/ thiosulfate using starch indicator
• if triiodate exceeds reducing capacity of thiosulfate, = indicator is blue (is clear before this)
• if smaller than amount of thiosulfate = indicator
• basically, I3 (triiodate) excess reacts with starch = BLUE COMPLEX

amoxicillin

• saw procedure for quantification of beta-lactam antibiotics (ensure quality of pharmaceutical product)
• studied if paper test card analysis could be useful for quality testing in low resource settings where analytical chem labs are rare

results of back titration of amoxicillin

• test card response showed good differentiation
• shows that cards could be used to detect antibiotics that contain 83% labeled API

analyte?

IODATE (IO3-)

matrix?

aqueous

is the method developed in the study quantitative or qualitative?

BOTH: QUANTITATIVE AND QUALITATIVE

• Qual - Color change
• Quant - Intensity of color using computer software

challenge of using PADs

reagents stored in paper substrate must be compatible + stable for long periods of time

what real world topic is the method developed aimed to address?

/ motivation for the method creation?

/ what they wanted to improve on?

to address analytical needs for salt producers

salt usually fortified with potassium iodate

many salt producers do not have access to a titration lab to assay the iodate

this test card (PAD) is designed to carry out the entire analytical process

how does the indicator in the assay work?

IT IS A STARCH

• fortified salt w/ iodate in model system to carry out the analytical task of measuring iodine in salt
• redox titration
• excess iodide + iodate + acid = triiodide
• then titratef w/ thiosulfate using starch indicator
• if triiodate exceeds reducing capacity of thiosulfate, = indicator is blue
• if smaller than amount of thiosulfate = indicator
• basically, I3 (triiodate) excess reacts with starch = BLUE COMPLEX

what is the endpoint in the back titration

and difference of PAD vs glassware titration

endpoint is when all S2O3 have reacted

Pad:

• endpoint = BLUE
• before eq. point (not all S2O3 reacted) = uncolored

glassware titration:

• endpoint = clear

can the assay be used for various other redox-active analytes?

YES

the device is VERSATILE

ex: quantified beta-lactam antibiotics via iodometric back titration

beta-lactam back titration

antibiotics quantified

degraded in base -> redox active thiol -> acidified + known amount excess triiodide added to oxidize thiol

unreacted triiodide is back titrated w/ thiosulfate

what is the STEM method used?

surface tension enabled mixing (STEM)

the samples and reagents are confined to the reaction area by the solution meniscus and the wide wax barriers

they are mixed by gently shaking

liquid applied to rxn area - forms dome confined by the solution meniscus. Reagents stored in the 5 loading zones dissolve + mix

how is titration done on the test cards?

zones and each reaction area are loaded with different amounts of sodium thiosulfate, excess potassium iodide, tosic acid, and starch indicator

can load up to 5 reagents on the card

where are the positive and negative controls?

they are designated reaction areas

• there are different reaction areas that act as positive or negative control

what is different about the card's end point vs a traditional titration?

traditional: has sharp end points

card: no color unless the amount of iodate in solution overwhelms the amount of thiosulfate -> then iodate content in the reaction area cause inc color production from the indicator until the response is saturated

limit test

test to make sure iodine content is below allowed value for edible salt

limit amount in salt for consumption is 15-30 ppm ish

square 11 labeled ">30" on the test card changes color = sample is over iodized = more iodate than allowed

what is measured on the test card?

ppm I

(iodine atoms/L solution)

see how it is the analyte?

ultimately measures 2 things

1. colorimetric quantificatino assay (the titration)
2. limit tests (determine if too much iodine for consumption)

diff parts of the card

1. negative control
   • only acid/starch
   • no response
2. limit test for iodide
3. quantify iodate in range of 0-4 ppm
4. quantify iodate in range of 0-4 ppm
5. quantify iodate in range of 0-4 ppm
6. quantify in range of 3-7 ppm
7. quantify in range of 3-7 ppm
8. quantify in range of 3-7 ppm
9. 7-11 ppm
10. 11-15 ppm
11. limit test for iodate in conc. greater than 30 ppm I
12. positive control

why was a lightbox used

bc quantification by color measurement is challenging if there are variations of images captured in the sun, shade, etc

light box has consistent illumination

blue color measured by what?

computer image analysis

what did the results of computer image analysis of the blue color on the test cards show?

sigmoidal increase in color with increasing iodate concentration

how many measurements were taken?

triplicate measurements taken to contribute to precision

more sensitive = bigger slope

systematic error

• camera software - adjusts exposure/coloration of image = lowers apparent iodine concentration
  • manual exposure could avoid this issue
• systematic underestimation of iodine concentration at high iodine concentrations - main source of inaccuracy

computer reading of cards advantages/disadvantages

• visual analysis inc usability in low resource settings
• using phone camera allows easy data collection + have in centralized network = more accessible

con

• visual analysis introduce human error in read/record results

how do different water sources and storage affect response?

• tap w/ high mineral water
  • 8% error
• lake water
  • 17% error

current methods for measurement of iodine in salt as well as their pros/cons

(why theyre good but also why they are not ideal for the conditions being addressed (address an analytical need of salt producers in the developing world, no access to a titration lab to perform the iodometric titrations)

• glassware titration
  • good precision/accuracy if done in lab + professionally
  • in field conditions in dev world settings -> accuracy/precision are compromised by
    • impure reagents
    • lack of calibration/service for analytical balances
    • overconcentrated titrant
• rapid test kits (RTKs)
  • inexpensive
  • but external validation study by WHO showed that tests are not reliable
• spectroscopic test kits
  • good metrics
  • but limited quantification range
  • but require purchase of specialized reader + kits of the chemicals used

saltPAD not most accuracy (middle), and least precise of the options, BUT is cheapest (for sure) and technical expertise needed is low

• sacrifices accuracy and precision for the sake of low cost and expertise needed, making it a good choice for settings with low ability to obtain and run the other methods discussed

why is back titration of amoxicillin done?

Wanted to address whether the paper test card developed could be useful in quality testing in low resource areas (useful for testing besides the iodate assay it was developed for)

to ensure antibiotic quality

places of low resources have substandard antibiotics + need to be checked for quality

ppm solution and salt and must convert to iodate

ppm I

= mg iodine atoms / L solution

Must convert to iodate first

Mg iodine mg/kg salt

Salt water soln – mg/L

How do LOD and LOQ used compare to the ones we used in class?

• LOD = 0.8 ppm I, defined by 3SD/m
• LLOQ = what we call the limit of quantification
  • they call it lower limit of quantification
  • defined same as us, as 10SD/m
  • 2.4 ppm I

eq 1 is about what?

precision calculated btw the 2 interpreters (x1 and x2)

eq2 is about what?

calc accuracy (real vs measured)

what is robustness and how did it play into the study?

if something is robust it means that external stimuli will not easily affect it. that is why the experimenters put the PAD in extreme conditions and show that it still works. I think this is also important in low resource settings as the storing conditions for the PADs might not be optimal.

• aging in convection oven
• lake wager vs hard water

why are they trying to find the analyte? (iodate)

bc they add it to fortify salts, and want to measure it in salts

why is the assay being made?

because they wanted to establish an assay that can be used in low resource areas, that can be done without chemistry equipment, electricity, etc. Iodate and beta-lactam are model systems, and they are trying to detect tests that can test various analytes in low resource areas

PAD

paper analytic device

device developed

ASSURED

• ASSURED (affordable, sensitive, specific, user friendly, rapid and robust, equipment free, deliverable to the end user) criteria to assess devices meant for use in low resource settings
• considered in design
• reasons how/why:
  • complete process = 0.09$ + $.5 labor + could be decreased
  • paper test card performs iodine analysis w/ accuracy + precision that rivals other technology used in salt fortification plants in the developing world + can perform iodometric back titration w/ to detect substandard antibiotics sufficient accuracy
  • takes 5 mins
  • test cards can withstand a lot of conditions
  • test cards can be analyzed using computer image processing of mobile phone photographs -> mobile phone network for data collection + archiving

why iodate?

dont give good reason, just chose as model system to detect an analyte

did same w/ beta lactam

just trying to create a test for low resource areas

how the assay works

separate into quadrants so reagents dont react - separate

sample in the middle

reaction steps

1. cs iodide (I-), dont know how much iodate (IO3-) so flood so it runs out = I3-

• excess I- in soln

2. titrate I3- with S2o3-

3. starch added

their spiking vs ours

their spiking is ???

ours is

• adding a known concentration of the analyte (spike), to an unknown concentration analyte in a sample, and determining the original unknown from the response (signal, intensity, etc)

the titrant is

I3- (triiodide)

pH meter

as ionic strength increases, the H+ is inhibited from reaching the pH meter and pH reads higher than it really is