The Portable LAMP application was written and developed in. JAVA in Android studio. This enabled images to be accessed via either the user's photo library or ...
A smartphone actuated isothermal amplification platform for rapid detection of nucleic acid assays Aashish Priye, Cameron S. Ball and Robert J. Meagher Sandia National Laboratories, Livermore, CA, USA
Integrated smartphone detection unit IPhone 5S
• The growing demand to deliver high-quality medical diagnostics in resource limited settings has created a drive to rapidly advance bio-analytical detection techniques.
IPhone Camera
Laser cut device for LAMP assay
• Here we demonstrate a completely automated and easy to use smart-phone based amplification and real time detection system for LAMP assay in a microfluidic chip.
• Complex • Bulky and heavy • Expensive (> $1,000)
• Ubiquitous • User friendly • Small footprint and ultra portable. • Low cost (< $100) • Low power operation
Isothermal hot plate
Bluetooth low energy (BLE) ATMEGA microcontroller
0
5
Onboard image analysis
Pixel matrix (green channel)
I green
[ R, G, B]1n [ R, G, B]mn
Positive Negative
100
1
Pre-LAMP
2
Post-LAMP
• The Portable LAMP application was written and developed in JAVA in Android studio. This enabled images to be accessed via either the user’s photo library or directly from the camera
[ R, G, B]1n [ R, G, B]mn
• The focus and exposure are locked before each image acquisition session ensuring constant lighting.
Gamma transformation (gRGB)
• The average, maximum and minimum RGBA pixel values for each image are calculated and can either be tabulated on the screen or plotted.
1/
[ R, G, B]s [ R, G, B] 255 255
• The selected analysis area is converted into bitmap image format stored as a mutable data set containing 4 bits per pixel corresponding to RGBA color space.
2.2
Acknowledgement End point detection
Real time detection
Red
60 Intensity (a.u.)
Stabilized reagents: LAMP assay reagents were vacuumdried using stabilizing formulations developed in partnership with Biomatrica. In accelerated aging tests, reagents were stable when stored for >3 weeks at 40 °C. This is expected to reduce dependence on cold chain for forward deployed assays.
[ R, G, B]11 Is [ R, G , B ] m1
Analysis area
200
0
RGB bitmat data
Bright signals: ROX fluorescence visualized with a green LED flashlight and red plastic film “filter”
-
Extract region of interest
Loop mediated isothermal amplification (LAMP)
0 [ R, G, B]11 1 0 [ R, G, B] m1
+
10
Raw image acquisition (sRGB)
Easily visualized endpoint: The SYTO intercalating dyes are compatible with closed-tube detection in LAMP, but are nonspecific, and offer ~3-fold difference in brightness between positive and negative reactions. Our novel technique allows >10X difference in brightness and can be visualized with an LED flashlight and colored plastic filter.
Visit Cameron Ball’s Poster for details
App for post processing
t (min)
West Nile Virus (WNV) RT-LAMP
• Easy integration with no external actuation. • Tunable opening pressure
Assembled device
Temperature profile
80 70 60 50 40 30 20
Laser cut process enables simple layer by layer assembly
Temperature feedback control
Temperature = 65 ºC
T (°C)
Smartphone detector
3D printed dark box
LED excitation source
• However, detection of amplified products via LAMP generally requires extra peripheral devices for post analytical fluorescence based optical detection (fluorometer) which makes the device bulky and increases the cost and complexity of the system.
Commercial detector
Bluetooth connectivity
Emission Filter
• Isothermal nucleic acid amplification tests such as LAMP (Loop mediated isothermal amplification) offer good sensitivity and specificity and eliminates the need for a thermal cycler, making it more suitable for point of care applications than the polymerase chain reaction (PCR).
Passive check valve assembly for LAMP assay
Intensity (a.u.)
Background and significance
40
Min
I avg
n
m
0
0
I
Max
Blue Avg
n,m
I
n.m Normali ze
Pre-LAMP
Four parameter sigmoid curve fitting (Marquardt-Levenberg algorithm)
20 0
Green
Post-LAMP
Data
I max
1 e
( t t1/2 )/ k
Io
• Funded by Sandia Laboratory Directed Research and Development (LDRD). Viral RNA was provided by Dr. Sarah Wheeler and Dr. Lark Coffey at the University of CaliforniaDavis Center for Vectorborne Diseases (CVEC). Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under Contract DE-AC04-94AL85000
