I spent some time yesterday thinking about whether we could do optical detection of DNA (particularly some variant of the DNA melting lab from MIT—see also the Fall 2008 class handouts). I noted in the 2008 handouts that they were using a blue LED array driven by a 0.29A current source (made from an LM317T voltage regulator, a rather inefficient method). The wiki page uses a regulated 5V supply and a 25Ω series resistor, which would be around 60–70mA for a typical forward drop of 3.2–3.5V in a blue LED. That’s still a pretty powerful light source for an LED. They say they are using LZ1-00B200, which has a 3.6V forward voltage, but can handle a full amp of current, so is more LED than is needed.
We can get a blue LED for under $2 that can handle 50mA continuously (LTL911CBKS5), though it has a forward voltage of typically 4.3V. In surface mount for $1.14, we could get MLEBLU-A1-0000-000T01, which has a dominant wavelength of 465–485nm (depending on bin code) and a luminous flux of 10.7 lm at 150mA (forward voltage 3.2V). I estimate the LED MIT mentions produces about 6lm at that current. The expensive part of the illumination is not the LED, but the focusing lenses to concentrate the light and the optical filtering needed to keep the excitation wavelength from being detected by the photodiode.
I was thinking that it would be cool to use a laser as an excitation source, rather than an LED, since then no lenses or filter would be needed on the source—just a blocking filter on the photoreceptor. Unfortunately, blue lasers are very expensive. What are cheap are the blue-violet lasers at 405nm, since the laser diodes are made in quantity for BluRay players. (Amazon has 405nm laser pointers for under $10 with shipping.) Unfortunately the usual fluorescent dyes used for DNA melting measurements (SYBR Green, LC Green Plus, EvaGreen) are not excited at 405nm, and need excitation wavelengths in the range 440nm–470nm). I’ve been wondering whether one of the 405nm-sensitive dyes used in flow cytometry (like Sytox Blue dead cell stain) could be used. But I’ve not found a double-stranded DNA dye for sale that is easily excited at 405nm (even Sytox Blue is way down in sensitivity from its peak), so laser excitation seems to be out—the excitation wavelengths needed for standard dyes require fairly expensive lasers. The benchtop lasers usually used in labs and flow cytometry equipment are priced in the “if-you-have-to-ask” price range. Buying enough copies for a student lab is more than this lab is worth.
I still don’t see a way to make the DNA melting curve project work within our course. Even MIT gives up half a semester to this lab, and we don’t have that much time (nor that caliber of students, on average).
I want the students to learn to solder (at least through-hole parts, not necessarily surface-mount). I don’t want to do the traditional blinky-light soldering practice, so I’ve been looking for a place in the course where it makes sense to require soldering, rather than wiring up a breadboard.
Breadboards have problems with loose wires, so the more complex the circuit, the more problems a breadboard causes. Breadboards also have problems connecting to wires that have to leave the breadboard—particularly wires to moving objects. This suggests that the EKG/EMG circuit would be the most appropriate as a soldering project, as it is fairly complicated and the long wires to the Ag/AgCl gel electrodes can cause a lot of problems with loose connections (my first check on debugging is to wiggle the header pins for those wires).
But I want the students to be doing some designing for the EKG circuit, not just soldering up a predetermined circuit, so I’m thinking of designing an instrumentation-amp protoboard, which has an ina126 instrumentation amp and an MCP6002 dual op amp chip, with power pins wired up and a place for the Rgain resistor and bypass capacitors, but everything else in a breadboard-like configuration, so that resistors, capacitors, and jumper wires could be added. Off-board connections could be done with screw terminals to make sturdy connections.
My son and I came up with the further idea of adding an optional LED output, to make a blinky-light-EKG device. I think that the approximately 1.5V, 30msec pulse that I was seeing for the R segment of the EKG would be enough to make a visible flash—I’ll have to try it out on my breadboard. I tried it today, but I was only seeing 0.5V pulses today (poorer contact with the electrodes?), and I had to raise the 3.9kΩ feedback resistor to 10kΩ to increase the gain of the final stage., which was enough to get weak flashes from an LED with a 100Ω series resistor. Because the op amp has limited output current (±23mA short circuit), I felt it fairly safe to put the LED directly between the op amp output and the Vref signal, which gives a good flash even with a green LED.
The lower voltage that I got this time (until I raised the gain) makes it clear that if I do make an EKG protoboard, it should have room for some trim pots for adjusting the final gain.
Filed under: Circuits course Tagged: Arduino, bioengineering, blinking light, circuits, course design, DNA melting, ECG, EKG, electrocardiogram, instrumentation amplifier, laser, op amp, pulse, violet lasers