Methods and systems for determining spatial patterns of biological targets in a sample

A method of determining a location of a target polynucleotide and a target protein in a sample comprising:

• (a) delivering a binding moiety-oligonucleotide conjugate to the sample, wherein the binding moiety-oligonucleotide conjugate comprises:
• (i) a binding moiety that specifically binds to the target protein, and (ii) an oligonucleotide conjugated to the binding moiety;
• (b) attaching the sample to a first microfluidic device having multiple first addressing channels, wherein a first addressing channel identifies a first area in the sample;
• (c) delivering a first probe through the first addressing channel to the first area in the sample, wherein the first probe comprises (i) a sequence substantially complementary to at least a portion of the target polynucleotide, (ii) a first address tag that identifies the first area in the sample, and (iii) a first ligation region;
• (d) delivering a second probe through the first addressing channel to the first area in the sample, wherein the second probe comprises (i) a sequence substantially complementary to at least a portion of the oligonucleotide conjugated to the binding moiety, (ii) the first address tag, and (iii) a second ligation region;
• (e) attaching the sample to a second microfluidic device having multiple second addressing channels, wherein a second addressing channel identifies in the sample a second area that intersects with the first area;
• (f) delivering a third probe through the second addressing channel to the second area in the sample, wherein the third probe comprises:
• (i) a second address tag that identifies the second area in the sample and (ii) a third ligation region, wherein the third probe is coupled to the first probe through ligation between the first ligation region and the third ligation region at an intersection between the first area and the second area, and the first address tag and the second address tag are used to identify the location of the target polynucleotide at the intersection in the sample;
• and
• (g) delivering a fourth probe through the second addressing channel to the second area in the sample, wherein the fourth probe comprises:
• (i) the second address tag, and (ii) a fourth ligation region, wherein the fourth probe is coupled to the second probe through ligation between the second ligation region and the fourth ligation region at an intersection between the first area and the second area, and the first address tag and the second address tag are used to identify the location of the target protein at the intersection in the sample.

A method of determining a location of a target protein in a sample, the method comprising:

• (a) delivering a first probe for the target protein to multiple locations in the sample, the first probe comprising (i) a binding moiety capable of specifically binding to the target protein, and (ii) an oligonucleotide conjugated to the binding moiety;
• (b) delivering a second probe to a first location in the sample, wherein the second probe comprises:
• (i) a sequence substantially complementary to a portion of the oligonucleotide of step (a), (ii) a first address tag that identifies the first location in the sample, and (iii) a first ligation region;
• and
• (c) delivering a third probe to a second location in the sample, wherein the third probe comprises (i) a second address tag that identifies the second location in the sample to which the third probe is delivered, and (ii) a second ligation region, wherein the second probe is coupled with the third probe through ligation between the first ligation region and second ligation region at an intersection between the first and second locations in the sample, and the first address tag and the second address tag are used to determine the location of the target protein at the intersection.