Problema with Arduino PCR THERMAL CYCLER PROGRAM

amable23 · May 11, 2021, 5:49pm

Hello,

We have built an Arduino PCR Thermal Cycler with an Arduino Nano, using a Thermocoupler K, a fan and resistors as heaters. When we start the machine, it works, but heats until 94 grades centigrades approximately and then starts the fan and never stops again, it does not restart to heat again in order to continue annealing and extension, and then denaturation, annealing and extension and so on.

Attached I send you the program (pcrssv.ino) that includes the library MAX31855 if anyone can help to find if there is a problem with any conditional or other part of the code.

#include <Adafruit_MAX31855.h>

/*

DIY Thermal Cycler for PCR (Summer School Reduced Version)
PCR code for denaturing, annealing and extending DNA cycles.
iDP (duilio.peroni@gmail.com) may 2018
Derived from: Stacey Kuznetsov (stace@cmu.edu) and Matt Mancuso (mcmancuso@gmail.com) July 2012
V 1.0 01/06/2018
Arduino based harware
- K-type thermocouple sensor with MAX6675 A/D converter
- 2x 50W resistor heater
- 12V fan cooler
- Start/stop button (internal pullup -> active-low signal)
- CSV remote log
PCR Cycle

       |              N x Cycles          |

 |  H  |   D   | C |    A    |H|   eX  |H |C|

Td _ _ _ _ _________ _ _ _ _ _ _ _ _ _ _ _ _ _ |

 |    /|       |\  |         | |       | /| |

Te _ _ _ /_ _ _ _ |_|_ _ _ _ _ ______/||

 |  /  |       |  \|         |/|       |\ | |

Ta _ _ /_ | _ _ | _________/|_ _ _ |||

 |/    |       |   |         | |       |  \ |

Tr / _ _ | _ _ | _ | _ _ _ _ || _ _ | |__

 |     |       |   |         | |       |  | |

CSV log format:
<total_time>;<cycle_number>;;<phase_time>;;
<total_time> :: HH:MM:SS
<cycle_number> :: 1..NUM_CYCLES
:: I=Idle, H=heating, C=cooling, D=denaturation, A=annealing, E=extension, F= fault
<phase_time> :: HH:MM:SS
:: tt.tt (°C)
:: 0=ok, 1=operator stop, 2=sensor not working, 3=temp already over setpoint, 4=temp not stable

*/
//libraries
//#include <max6675.h> //include library for K-junction thermocouple A/D converter

//hardware definition (based on Arduino UNO/Leonardo/Micro/Nano or compatibles)
//input
#define START_STOP_PIN 8 //start/stop cycle pushbutton
#define THERMO_DO 4 //thermocouple data pin
#define THERMO_CS 5 //thermocouple select pin
#define THERMO_CLK 6 //thermocouple clock pin
//output
#define HEAT_PIN 7 //heater resistor relay output
#define FAN_PIN 9 //fan driver output

//PCR parameters

//cycles numbers
#define NUM_CYCLES 32 //number of cycles of PCR sequence

//temperatures
#define DENATURE_TEMP 94.0 //denaturation phase temperature
#define ANNEALING_TEMP 65.0 //annealing phase temperature
#define EXTENSION_TEMP 72.0 //extension phase temperature
#define FAN_THRESHOLD_TEMP 0.75 //fan threshold temperature (in order to avoid underruning temperature in cooling)
#define SETPOINT_THRESHOLD_TEMP 0.5 //setpoint threshold temperature (in order stabilize setpoit holding)
#define STABILITY_THRESHOLD_TEMP 1.25 //max deviation from setpoint
#define APPROACH_THRESHOLD_TEMP 2.0 //approaching temp threshold in heating

//other heating parameters
#define MAX_PULSE_DURATION 650.0 //max duration of an heating pulse (mS)
#define PULSE_DURATION_MULT 600.0 //multiplication coefficient to get pulse duration proportional to temperature gap (adimensional)
#define MIN_PULSE_DURATION 80.0 //min duration of an heating pulse when approaching to setpoit (mS)
#define OVERHEAT_TEST 30 //number of iteration before each overheat test (adimensional)
#define OVERHEAT_TIME 250 //overheat wait pulse (no heating, mS)

//times
#define DENATURE_TIME 33000 //denaturation phase duration
#define ANNEALING_TIME 333000 //annealing phase duration
#define EXTENSION_TIME 35000 //extension phase duration
#define INITIAL_DENATURE_TIME 300000 //first denaturation phase duration
#define FINAL_EXTENSION_TIME 600000 //last extension phase duration
#define COOL_SAMPLE_TIME 300 //temperature sample time during cooling phase
#define HOLD_PULSE_DURATION 210 //hold temperature pulse duration
#define HOLD_PAUSE_DURATION 90 //hold temperature pulse duration

//safety params (°C)
#define ROOM_TEMP 18 //room temperature setpoit (to check for sensor failure)
#define MAX_ALLOWED_TEMP 100 //max allowed temperature (for safety purpose)
#define MAX_HEAT_INCREASE 2.5 //max slope of rising temperature (for safety purpose)
#define MAX_ALLOWED_TEMP_TIME 1000 //max allowed temp sample time
#define MAX_HEAT_INCREASE_TIME 1000 //max heat increase sample time

//Phases states
#define IDLE 0 //waiting for a cycle
#define HEATING 1 //heating rising temperature ramp
#define COOLING 2 //cooling falling temperature ramp
#define DENATURATION 3 //denaturation phase
#define ANNEALING 4 //annealing phase
#define EXTENSION 5 //extension phase
#define FAULT 6 //fault status

//Phases names
char phases[7][5]={"IDLE","HEAT","COOL","DEN ","ANN ","EXT ","FLT"};

//Utility function prototypes
void runPCR(void);
boolean heatUp(double setpoint);
boolean coolDown(double setpoint, int maxTimeout = COOL_SAMPLE_TIME);
boolean holdConstantTemp(long duration, double setpoint);
void log(boolean force=false);
boolean buttonPressed(void);
boolean serialEvent(void);

//global variables
int currInButton; //current loop status of start/stop pushbutton
int prevInButton; //previous loop status of start/stop pushbutton
unsigned long startCycleTime; //cycle start time;
unsigned long startPhaseTime; //phase start time;
unsigned long timeCounter; //
int currCycle; //current cycle value
int currPhase; //current phase value
double currTemp; //current temperature value
double prevTemp; //previuous temperature value
char faultCode='0'; //fault code
//temperature device wrapper
Adafruit_MAX31855 thermocouple(THERMO_CLK, THERMO_CS, THERMO_DO);

//setup: run once at startup
void setup() {
//init console
Serial.begin(250000);

//configure I/O
pinMode(START_STOP_PIN,INPUT_PULLUP);
pinMode(HEAT_PIN,OUTPUT);
pinMode(FAN_PIN,OUTPUT);
//set I/O initial status
currPhase=IDLE;
digitalWrite(HEAT_PIN,LOW);
digitalWrite(FAN_PIN,LOW);
}

//loop: run repeatedly forever
void loop() {
//check for start button pressed (HIGH to LOW transition)
currTemp = thermocouple.readCelsius();
Serial.println(currTemp);
if(buttonPressed()){
//run a full PCR cycle on start event
runPCR();
}
//check for serial command ('s' or 'S')
if(serialEvent()){
//run a full PCR cycle on serial command
runPCR();
}
}

//runPCR: run a full PCR sequence
//repeat NUM_CYCLE times the PCR sequence:
// Heating to denaturation temp
// Denaturation phase
// Cooling to annealing temp
// Annealing phase
// Heating to extension temp
// Extension phase
// at the end of cycle cool to room temp

void runPCR(void){
boolean ris; //phase result

//set start time
startCycleTime=millis();
timeCounter=0;
faultCode='0';

//repeat for NUM_CYCLES cycles
for(currCycle=0;currCycle<NUM_CYCLES;currCycle++){

//heatup to denature temperature
currPhase=HEATING;
ris=heatUp(DENATURE_TEMP);
if(ris==false){
  //heating fault or stop button: stop sequence
  currCycle = NUM_CYCLES;
  currPhase=FAULT;
  log();
  break;
}

//denaturarion phase
currPhase=DENATURATION;
if(currCycle == 0) {
  //first cycle: special denature time
  ris=holdConstantTemp(INITIAL_DENATURE_TIME, DENATURE_TEMP);
} 
else {
  //no first cycle: standard denature time
  ris=holdConstantTemp(DENATURE_TIME, DENATURE_TEMP);
}
if(ris==false){
  //denaturation fault or stop button: stop sequence
  currCycle = NUM_CYCLES;
  currPhase=FAULT;
  log();
  break;
}

//cool to annealing temperature
currPhase=COOLING;
ris=coolDown(ANNEALING_TEMP);
if(ris==false){
  //cooling fault or stop button: stop sequence
  currCycle = NUM_CYCLES;
  currPhase=FAULT;
  log();
  break;
}

//annealing phase
currPhase=ANNEALING;
ris=holdConstantTemp(ANNEALING_TIME, ANNEALING_TEMP);
if(ris==false){
  //annealing fault or stop button: stop sequence
  currCycle = NUM_CYCLES;
  currPhase=FAULT;
  log();
  break;
}

//heatup to extension temperature
currPhase=HEATING;
ris=heatUp((EXTENSION_TEMP));
if(ris==false){
  //heatup fault or stop button: stop sequence
  currCycle = NUM_CYCLES;
  currPhase=FAULT;
  log();
  break;
}

//extension phase phase
currPhase=EXTENSION;
if (currCycle<(NUM_CYCLES-1)) {
  //not last cycle: standard extension time
  ris=holdConstantTemp(EXTENSION_TIME, EXTENSION_TEMP);
} 
else {
  //last cycle: special extension time
  ris=holdConstantTemp(FINAL_EXTENSION_TIME, EXTENSION_TEMP);
}
if(ris==false){
  //extension fault or stop button: stop sequence
  currCycle = NUM_CYCLES;
  currPhase=FAULT;
  log();
  break;
}

}

//final cooling (indefinite time)
digitalWrite(FAN_PIN,HIGH);
if(currPhase!=FAULT) { //normal termination
currPhase=IDLE;
}
log(true);
}

//heatUp: heat up to maxTemp temperature setpoint
//return true if maxTemp reached, false if fault (sensor not working, temp not reached, overheat)
//
boolean heatUp(double setpoint){
boolean ris=false;
int curIteration = 0;
digitalWrite(FAN_PIN,LOW);
startPhaseTime=millis();
prevTemp=thermocouple.readCelsius();
currTemp=prevTemp;
log();
//test if sensor working: if reading under ROOM_TEMP we assume sensor not working and stop the sequence
if (currTemp < ROOM_TEMP) { //sensor not working
faultCode='2';
ris=false;
}
else if(currTemp > setpoint){ //over setpoint entering heating
faultCode='3';
ris=false;
}
else { //sensor ok: execute heating rise ramp
//repeat indefinitely while temperature under setpoint
while (currTemp < setpoint) {
//check stop button
if(buttonPressed()){
faultCode='1';
ris=false;
break;
}
curIteration++;
int pulseDuration = min(MAX_PULSE_DURATION, ((PULSE_DURATION_MULT *(setpoint-currTemp))+ MIN_PULSE_DURATION)); // as we approach desired temp, heat up slower
digitalWrite(HEAT_PIN, HIGH);
delay(pulseDuration);
currTemp=thermocouple.readCelsius();

  log();
  digitalWrite(HEAT_PIN, LOW);
  //check if setpoint reached
  if(currTemp >= setpoint) { //checkpoint reached: stop rise ramp with success
    faultCode='0';
    ris=true;
    break;
  }
  //approaching to setpoint insert a little wait between heat pulses to avoid overheat

// if((setpoint-currTemp) < APPROACH_THRESHOLD_TEMP || curIteration % OVERHEAT_TEST == 0) {
if((setpoint-currTemp) < APPROACH_THRESHOLD_TEMP ) {
do {
prevTemp = currTemp;
delay(OVERHEAT_TIME);
currTemp=thermocouple.readCelsius();
log();
} while(currTemp > prevTemp);
}
//check again if setpoint reached
if(currTemp >= setpoint){ //checkpoint reached: stop rise ramp with success
faultCode='0';
ris=true;
break;
}
//check each two iteration if setpoint not stable
if ((curIteration%2) == 0) { //each two iterations
if(currTemp < (prevTemp-STABILITY_THRESHOLD_TEMP)) { //setpoint not stable stop rise ramp with failure
faultCode='4';
ris=false;
break;
}
}
else {
prevTemp = currTemp;
}
//check if rise ramp too fast
while ((currTemp-prevTemp) >= MAX_HEAT_INCREASE) { //rise ramp too fast: wait for slope reduction
prevTemp = currTemp;
delay(MAX_HEAT_INCREASE_TIME);
currTemp=thermocouple.readCelsius();
log();
}
//check for max temperature
while(currTemp >= MAX_ALLOWED_TEMP) { //temperature too high: wait for temperature reduction
delay(MAX_ALLOWED_TEMP_TIME);
currTemp=thermocouple.readCelsius();
log();
}
}
}
return ris;
}

//coolDown: Cool down to the desired temperature by turning on the fan.
// setpoint -> Temperature we want to cool off to
// maxTimeout -> how often we poll the thermocouple (300ms is good)
boolean coolDown(double setpoint, int maxTimeout = COOL_SAMPLE_TIME){
boolean ris=true;
startPhaseTime=millis();
currTemp=thermocouple.readCelsius();
log();
while (currTemp > setpoint+FAN_THRESHOLD_TEMP) {
digitalWrite(FAN_PIN, HIGH);
delay(maxTimeout);
currTemp=thermocouple.readCelsius();
log();
//check stop button
if(buttonPressed()){
faultCode='1';
ris=false;
break;
}
}
digitalWrite(FAN_PIN, LOW);
return ris;
}

//holdConstantTemp: Try to stay close to the desired temperature by making micro adjustments to the
// resistors and fan. Assumes that the current temperature is already very close to the setpoint.
// setpoint -> desired temperature
// duration -> how long we should keep the temperature (in ms)

boolean holdConstantTemp(long duration, double setpoint) {
boolean ris=true;
//setup phase timer
startPhaseTime=millis();
unsigned long holdTimeElapsed = 0;

//hold temperature to setpoint for duration time
while (holdTimeElapsed < duration) {

//check stop button
if(buttonPressed()){
  faultCode='1';
  ris=false;
  break;
}

//hold temperature
currTemp = thermocouple.readCelsius();
log();
if (currTemp < setpoint) { //under setpoint: heat up 
  digitalWrite(HEAT_PIN, HIGH);
  delay(HOLD_PULSE_DURATION);
  digitalWrite(HEAT_PIN, LOW);
} 
else if (currTemp > (setpoint+SETPOINT_THRESHOLD_TEMP)) { //over setpoint: cool down
  digitalWrite(FAN_PIN, HIGH);
  delay(HOLD_PULSE_DURATION);
  digitalWrite(FAN_PIN, LOW);
}
else {
  delay(HOLD_PULSE_DURATION);      
}
delay(HOLD_PAUSE_DURATION);
holdTimeElapsed = millis() - startPhaseTime;

}
return ris;
}

//log: send a CSV data line to console & LCD diagnostic
//CSV log format:
//<total_time>;<cycle_number>;;<phase_time>;;

void log(boolean force=false){
unsigned long cycleTime=millis()-startCycleTime;
unsigned long phaseTime=millis()-startPhaseTime;
char buffer[9];
//cycle time in HH:MM:SS format
unsigned long ts=cycleTime/1000; //time in seconds
int h = ts/3600; //hours
int t = ts % 3600; //remaining seconds
int m = t/60; //minutes
int s = t%60; //seconds
if((ts>timeCounter)||(force==true)) { //second elapsed: print log
timeCounter=ts;
sprintf(buffer,"%02d:%02d:%02d",h,m,s);
Serial.print(buffer);
Serial.print(";");
//cycle number (startig from 1)
sprintf(buffer,"%02d",currCycle+1);
Serial.print(buffer);
Serial.print(";");
//cycle phase (Idle,Heating,Cooling,Denature,Annealig,Extension,Fault)
Serial.write(phases[currPhase][0]);
Serial.print(";");
//phase time in HH:MM:SS format
ts=phaseTime/1000; //time in seconds
h = ts/3600; //hours
t = ts % 3600;
m = t/60; //minutes
s = t%60; //seconds
sprintf(buffer,"%02d:%02d",m,s);
Serial.print(buffer);
Serial.print(";");
//temperature in tt.tt (°C) format
int iPart=(int)currTemp;
int dPart=(int)((currTemp - (double)iPart) * 100);
sprintf(buffer, "%03d.%02d", iPart, dPart);
Serial.print(buffer);
Serial.print(";");
Serial.write(faultCode);
Serial.println();
}
}

//buttonPressed: check for falling edge of start/stop button
// returns true: button pressed, false: button not pressed
boolean buttonPressed(void){
boolean ris=false;
currInButton=digitalRead(START_STOP_PIN);
if((currInButton!=prevInButton)&&(currInButton==LOW)){ //falling edge
ris=true;
}
prevInButton=currInButton;
return ris;
}

//serialEvent: check for falling command from serial
// returns true: command from serial
boolean serialEvent(void){
boolean ris=false;
if(Serial.available()){
int rx=Serial.read();
if((rx=='s')||(rx=='S')){
if((currPhase==IDLE)||(currPhase==FAULT)) {
ris=true;
}
}
}
return ris;
}

Thanks in advance.

johnwasser · May 11, 2021, 9:50pm

What does the log say?

amable23 · May 12, 2021, 9:22am

Measure the temperature at 94 grades, starts to Cool and falls temperature at the fan does not stop. It is possible that if fan cannot decrease quickly the temperature to 65, may be the reason why the fan never stops?

johnwasser · May 12, 2021, 12:11pm

That's interesting. What does the log say?

system · September 9, 2021, 12:11pm

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