A Field-deployable Platform for Prognostic Hepatic Cancer Screening in Low-resource Settings

Challenge: Develop a point of need (PON) platform to screen at-risk populations for HCC. Solution: Combine membrane based assay technology with handheld spectrometric readout.

A Field-deployable Platform for Diagnostic Hepatocellular Carcinoma Screening in Low-resource Settings Jennifer H. Granger , Jill E. Shea , Courtney L. Scaife , and Marc D. Porter 1 Mongolia 40 Average MONGOLIA LAO PDR GAMBIA, THE EGYPT, ARAB REP. VIETNAM NORTH KOREA CHINA THAILAND CAMBODIA GUINEA SIERRA LEONE LIBERIA COTE D'IVORIE SOUTH KOREA GUATEMALA VANUATU BURKINA FASO BENIN GUINEA-BISSAU MAURITANIA RWANDA CABO VERDE TOGO SENEGAL NIGERIA PHILIPPINES SOLOMON ISLANDS HONDURAS PAPUA NEW GUINEA GHANA MYANMAR CONGO NICARAGUA SWAZILAND ARMENIA SINGAPORE JAPAN 0 Challenge: Develop a point of need (PON) platform to screen at-risk populations for HCC Raman Readout Strategy Solid Phase Extractions Fundamentals Nitrocellulose membrane modified with capture antibody •Nearly all diagnosed HCC cases in Mongolia are referred to the National Cancer Center of Mongolia (NCCM), located in the capital city of Ulaanbaatar. 60 20 •Patient survival is compounded by the lack of and geographic access to preventative care. Apply patient sample •Unfortunately, ~90% of patients seeking HCC treatment at NCCM are late stage (i.e. tumors >2cm) and are not eligible for resection or other curative treatments. Target biomarker is selectively captured Raman Response High Biomarker Concentration Medium Biomarker Concentration 1200 1350 Raman Shift / cm-1 1500 The height of this peak (measured in cts/s) is used to construct a calibration plot which is correlated to captured biomarker concentration. 10000 8000 Alphafetoprotein Lens-culinaris agglutinin binding Alphafetoprotein Des-GammaCarboxy Prothrombin Core Antibodies to Hepatitis B Virus Core Antibodies to Hepatitis C Virus Abbreviation AFP Function 4000 2000 fetal glycoprotein that is increased in HCC AFP-L3 fucosylated variant of AFP that has a high affinity to Lens culinaris produced by malignant hepatocytes DCP arises from an acquired defect in post-translational carboxylation of the prothrombin precursor in malignant hepatocytes HBV HCV small enveloped DNA virus that can lead to HCC small enveloped DNA virus that can lead to HCC 0 0 10 20 30 40 Marker Concentration (pg/mL) B&W Tek TacticID Handheld Effect of Blocking Agent on Performance Blocking Agent Digital Image of Membrane Raman Signature Raman Response 10 ng/mL AFP 15000 cts/s 1% Bovine Serum Albumin ≥10 ng/mL 2000 cts/s 100 µg/mL Casein membrane blocked 16000 14000 blank (serum) not detected membrane blocked 12000 10000 8000 6000 4000 2000 0 0 Phosphate Buffered Saline 0.1% Tween 20 >10% of total AFP (1 ng/mL) 15000 cts/s 10000 cts/s 2 4 6 8 10 12 Serum AFP Concentration (ng/mL) Calibration Plot for AFP using 1% BSA to block membranes LOD reduced to ~3 pg/mL Conclusions and Future Directions 0.1 µg/mL •A PON assay for AFP has been successfully developed for AFP with a LOD ~3 pg/mL, which is over 3000x lower than the proposed clinical value of 10 ng/mL. ≥10 ng/mL •We are actively working towards multiplexed detection of the markers in Table 1. Blank 5 ng/mL 1000 5 ng/mL •Embedded software and database for Yes/No answer •Multiplexed detection 6000 Table 1. Serum markers of HCC and Infectious Disease That Increase HCC Risk Biomarker •No refrigeration needed to store reagents 12000 Detection of Alphafetoprotein in Serum Target Levels for Risk Assessment •Minimal cost ($2/test) Low Biomarker Concentration 1050 After labeling, membrane is directly read using handheld Raman. Development of a HCC Marker Panel Membrane Technology + Raman Readout •No sample preparation; •Weight < 2 lbs (0.9 Kg) matrix agnostic •8 hr rechargeable battery •Sample-to-answer in •Ethernet or WiFi less than 2 minutes connectivity The Raman-active molecules on the gold nanoparticle labels give a characteristic peak at 1336 cm-1 (outlined in a blue dashed box (left)). Blank Gold nanoparticle labels with target-specific antibodies and Raman-active molecules are applied. Solution: Combine membrane based assay technology with handheld spectrometric readout Merits of µSPERR Peak Height (cts/s) 80 micro-Solid Phase Extraction with Raman Readout (µSPERR) SERS Intensity (cts s -1) 100 1, 3-6 Nano Institute of Utah, University of Utah Departments of 2Surgery, 3Chemistry, 4Engineering and 5Pathology Hepatocellular Carcinoma (HCC) in Mongolia Global Incidence of HCC 2 SERS Intensity / cts s-1 1 2 1200 1400 1600 Add mixture of gold nanoparticle labels 1800 Raman Shift (cm-1) Although a red color is clearly visible at high AFP concentrations (100 µg/mL), detection at levels of 0.1 µg/mL and below requires Raman readout. The calculated limit of detection (LOD) is ~50 ng/mL. Raman label 1 (AFP) Raman label 2 (AFP-L3) Raman label 3 (DCP) Raman label 4 (HBV/HCV) Apply a single patient sample This work was supported by the Kerith Foundation and Huntsman Cancer Institute.